Synergistic Enhancement of the Delivery of Nucleic Acids via Blended Formulations

ABSTRACT

Disclosed herein are pharmaceutical compositions that comprise “blends” of lipid nanoparticles and related methods of using such blended compositions to deliver polynucleotides to one or more target cells, tissues or organs. The blended compositions are generally characterized as being able to efficiently deliver polynucleotides to target cells and by their ability to enhance the expression of such polynucleotides and the production of functional proteins by target cells.

RELATED APPLICATIONS

This application is a divisional application of the U.S. Application No.16,157,050, filed on Oct. 10, 2018, which is a continuation of the U.S.application Ser. No. 14/775,818, filed on Sep. 14, 2015, now issued asU.S. Pat. No. 10,130,649, which is a National Stage Entry of theInternational Application PCT/US2014/028498, filed on Mar. 14, 2014,which claims priority to U.S. Provisional Application Ser. No.61/789,375 filed Mar. 15, 2013, the disclosure of which is herebyincorporated by reference.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

This instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 31, 2020, isnamed MRT-1101US3_ST25.txt and is 6,266 bytes in size. No new matter ishereby added.

BACKGROUND

The efficient delivery of nucleic acids to targeted cells and tissues,as well as the subsequent transfection of such nucleic acids into suchtargeted cells and tissues remains a technical challenge. For example,as a result of the size and charge of nucleic acids such as DNA and RNA,the ability to effectively and efficiently deliver such nucleic acids toand/or to transfect such targeted cells and tissues is often limited.

One approach to improve the delivery of nucleic acids andpolynucleotides to target cells and tissues has been the liposomalencapsulation of nucleic acid in lipids, and in particular cationiclipids. The electrostatic interaction of the cationic lipid with nucleicacids facilitates the formation of lipid-encapsulated nucleic acidparticles in size ranges which may be suitable for in vivoadministration. The positively charged cationic lipid on the outerparticle surfaces facilitates the interaction of the cationiclipid-based liposome with the negatively-charged cellular membranes,thereby promoting fusion of the liposome with the cellular membrane anddelivering the liposome and/or emptying the nucleic acid contents of theliposome intracellularly. Although several advantages of using liposomesto facilitate the delivery of therapeutic agents to target cells andtissues have been previously described, many problems still exist in invivo, ex vivo and in vitro applications. For example, many of thecationic lipids are generally toxic to the targeted cells, andaccordingly may be of limited use. Furthermore, liposomal carriers maynot be the most efficient means of delivering nucleic acids to targetcells or to subsequently transfect such cells.

Novel approaches and therapies are still needed to enhance the deliveryand/or transfection efficiencies of polynucleotides and nucleic acids,particularly those delivered in liposome delivery vehicles, such asencapsulated liposomal formulations. The development of new and improvedliposome delivery vehicles and liposomal formulations that demonstrateenhanced delivery and/or transfection efficiencies would further advancenucleic acid based therapies for the treatment of diseases, such as geneand mRNA silencing therapies, that may benefit from gene replacementtherapies, mRNA delivery therapies, and/or other therapies that includethe intracellular delivery of nucleic acids for the modulation of gene,protein and/or enzyme expression.

SUMMARY

The present invention is, in part, based on the unexpected discoverythat a blend of multiple non-identical lipid nanoparticlessynergistically enhances the expression of messenger RNA (mRNA)encapsulated within at least one of the lipid nanoparticles in vivo.This synergistic effect is observed across a wide variety of differentlipid nanoparticles blended in various ratios and via differentadministration routes. For example, in some cases, enhancements inprotein expression (e.g., evidenced by light output of a fireflyluciferase) ranged from about 1.5-fold to 30-fold increase as comparedto the additive total based on each individual nanoparticle. The synergyis also observed in both tissue specific and systemic expression ofmRNA. More surprisingly, this synergistic effect is not nucleic acidspecific. For example, a non-fluorescent mRNA can synergisticallyenhances the light production of a fluorescent mRNA. It is contemplatedthat this unexpected discovery of synergistic enhancement betweendifferent lipid formulations has significant implication in messengerRNA therapy because it allows equivalent therapeutic efficacy beachieved via administration of a significantly lower dose. The abilityto create a synergistic production of protein via lipid-basednanoparticle delivery of mRNA also permits a much greater therapeuticwindow for the treatment of a host of diseases and achieves equal orgreater efficacy while minimizing any adverse or toxic event. Thus, thepresent invention provides a safer and more potent messenger RNA therapyfor various diseases.

Among other things, disclosed herein are pharmaceutical compositionsthat comprise a blend of at least two lipid nanoparticles (e.g., a blendof a first lipid nanoparticle and a second lipid nanoparticle) andrelated methods of using such blended nanoparticle compositions. Incertain embodiments, at least one of the constituent lipid nanoparticlesthat comprises the blended lipid nanoparticle composition comprises(e.g., encapsulates) one or more polynucleotides (e.g., mRNA). Theblended lipid nanoparticle compositions described herein arecharacterized as being able to efficiently deliver the encapsulatedpolynucleotides to target cells, and are also characterized by theirability to improve the subsequent transfection of such encapsulatedpolynucleotides following contacting one or more of such target cells.The blended lipid nanoparticle compositions are also characterized bytheir ability to modulate or enhance (e.g., synergistically increase)the expression of the polynucleotides encapsulated therein by targetcells. In certain embodiments where the polynucleotides are mRNA, theblended lipid nanoparticle compositions are also characterized by theirability to enhance the production of polypeptides encoded by suchpolynucleotides.

As used herein, the term “blend”, “blended”, or grammatical equivalent,refers to a combination of two or more separate, non-identicalformulations. Typically, the two or more separate, non-identicalformulations are combined or blended into one composition, such as, asuspension, as depicted, for example, in FIG. 1. As used herein,non-identical formulations refer to formulations containing at least onedistinct lipid component. In some embodiments, non-identicalformulations suitable for blend contain at least one distinct cationiclipid component. The term “blend” as used herein is distinguishable fromthe terms “mix” or “mixture”, which are used herein to define a singleformulation containing multiple non-identical cationic/ionizable lipids,multiple non-identical helper lipids, and/or multiple non-identicalPEGylated lipids. In some embodiments, a “mix” formulation contains atleast two or more non-identical cationic/ionizable lipids. Typically, a“mix” formulation contains a single homogeneous population of lipidnanoparticles.

Certain embodiments relate to methods of expressing one or morepolynucleotides in one or more target cells. For example, where thepolynucleotides are mRNA encoding a functional protein, provided hereinare methods of enhancing the production and/or excretion of polypeptidesencoded by such polynucleotides by a target cell. Certain embodimentsrelate to methods of modulating the expression of one or morepolynucleotides or nucleic acids (e.g., a target nucleic acid) in one ormore target cells, using for example an antisense oligonucleotide. Suchmethods may comprise contacting the one or more target cells with apharmaceutical composition comprising a blend of at least two lipidnanoparticles (e.g., a first lipid nanoparticle and a second lipidnanoparticle), wherein such first and second lipid nanoparticles havedifferent lipid compositions (e.g., the first lipid compositioncomprises a different cationic lipid than the second lipid nanoparticlecomposition). In certain embodiments, at least one of the two or morelipid nanoparticles that comprise the blended lipid nanoparticlecomposition comprises one or more polynucleotides. For example, thefirst lipid nanoparticle may encapsulate one or more polynucleotides andthe second lipid nanoparticle may optionally encapsulate one or morepolynucleotides.

In certain embodiments, the blended first lipid nanoparticle and secondlipid nanoparticle comprise the same one or more polynucleotides,wherein the expression of the one or more polynucleotides by the targetcells following the administration (e.g., intravenously) of the blendedpharmaceutical composition to a subject exceeds the relative sum of theexpression of the one or more polynucleotides achieved by the firstlipid nanoparticle and the expression of the one or more polynucleotidesachieved by the second lipid nanoparticle when the first lipidnanoparticle and the second lipid nanoparticle are administered to thesubject independently of each other. For example, in certainembodiments, the expression of the one or more polynucleotides by thetarget cells following the administration (e.g., intravenously) of suchblended pharmaceutical composition to a subject may exceed the relativesum of the expression of the one or more polynucleotides achieved by thefirst lipid nanoparticle and the expression of the one or morepolynucleotides achieved by the second lipid nanoparticle when the firstlipid nanoparticle and the second lipid nanoparticle are independentlyadministered to the subject by at least about two-, five-, ten-,twelve-, fifteen-, twenty-, twenty-five-, thirty-, forty-, fifty-fold,or more.

In certain embodiments, the blended first lipid nanoparticle and secondlipid nanoparticle comprise the same one or more polynucleotides (e.g.,mRNA), wherein the production of one or more polypeptides or proteins(e.g., an enzyme) by the target cells following the administration(e.g., intravenously) of the blended pharmaceutical composition to asubject exceeds the relative sum of the production of the one or morepolypeptides or proteins (e.g., an enzyme) produced following thedelivery of such one or more polynucleotides achieved by the first lipidnanoparticle and the one or more polypeptides produced following thedelivery of the one or more polynucleotides achieved by the second lipidnanoparticle when the first lipid nanoparticle and the second lipidnanoparticle are administered to the subject independently of eachother. For example, in certain embodiments where the polynucleotidescomprise mRNA, the polypeptides produced following the delivery of suchpolynucleotides to the target cells following the administration (e.g.,intravenously) of such blended pharmaceutical composition to a subjectmay exceed the relative sum of the polypeptides produced following thedelivery of such polynucleotides achieved by the first lipidnanoparticle and the polypeptides produced following the delivery ofsuch polynucleotides achieved by the second lipid nanoparticle when thefirst lipid nanoparticle and the second lipid nanoparticle areindependently administered to the subject by at least about two-, five-,ten-, twelve-, fifteen-, twenty-, twenty-five-, thirty-, forty-, fifty-,sixty-, seventy-, eighty, ninety-, one-hundred-, five-hundred-, onethousand-fold, or more.

In another embodiment, only one of the two or more lipid nanoparticlesthat comprise the blended composition comprises or encapsulates apolynucleotide. For example, where the pharmaceutical compositionscomprises two blended lipid nanoparticles, only the first lipidnanoparticle comprises one or more polynucleotides while the secondlipid nanoparticle does not comprise a polynucleotide (i.e., the secondpolynucleotide is empty). In such an embodiment, following theadministration (e.g., intravenously) of the two blended first and secondlipid nanoparticles that comprise the pharmaceutical composition to thesubject, the production of one or more polypeptides or proteins encodedby the encapsulated polynucleotides by a target cell is enhancedrelative to the production of one or more polypeptides or proteinsobserved when the first lipid nanoparticle is administered to thesubject independently of the second lipid nanoparticle. For example, insuch an embodiment, the production of the one or more polypeptides orproteins by the target cells following the administration of suchblended pharmaceutical composition to a subject exceeds the productionof the one or more polypeptides or proteins when the first lipidnanoparticle is administered to the subject independently of the secondlipid nanoparticle by at least about two-, five-, ten-, twelve-,fifteen- or twenty-fold, twenty-five-, thirty-, forty-, fifty-,one-hundred-, five-hundred-, one thousand-fold or more.

Upon contacting one or more targeted cells with the blended lipidnanoparticle compositions disclosed herein (e.g., by intravenouslyadministering the blended pharmaceutical composition to a subject) oneor more of such target cells are transfected with and may express theone or more polynucleotides and/or enhance the production of one or morefunctional polypeptides or proteins encoded by such one or morepolynucleotides. In certain embodiments, contacting such target cellswith the blended lipid nanoparticles and pharmaceutical compositionssuch that the target cells are transfected by the encapsulated one ormore polynucleotides enhances (e.g., synergistically increases) theexpression of such polynucleotides and/or increases the production of afunctional protein or polypeptide product that may be useful in thetreatment of a disease or pathological condition (e.g., diseasesresulting from a protein or enzyme deficiency). In certain embodiments,upon or following transfection by the blended lipid nanoparticlecompositions described herein, the expression of the encapsulatedpolynucleotides and/or production of a functional polypeptide or proteinby one or more target cells is synergistically increased, and inparticular is synergistically increased relative to the expression ofthe polynucleotides and/or production of a the functional polypeptide orprotein that is observed when the constituent lipid nanoparticles thatcomprise the blended composition (e.g., the first lipid nanoparticle andthe second lipid nanoparticle) are administered independently of theother. The expression of the encapsulated polynucleotides that comprisethe blended composition (and/or where such polynucleotides comprisemRNA, the production of the polypeptides encoded by such encapsulatedpolynucleotides) may be increased, for example, by at least about two-,three-, four-, five-, six-, eight-, ten-, twelve-, fifteen-, twenty-,twenty-five-, thirty-, forty- or fifty-fold, or more relative to theexpression of polynucleotide (and/or the production of polypeptide wheresuch polynucleotide comprises mRNA) that is observed when theconstituent lipid nanoparticles that comprise the blended formulationare independently administered.

Also disclosed herein are methods of modulating or increasing theexpression of one or more polynucleotides and methods of increasing theproduction of one or more functional polypeptides or proteins in one ormore target cells (e.g., target cells of a subject to whom the blendedlipid nanoparticle compositions are administered). Such methods maycomprise the step of administering or otherwise contacting targetedcells or tissues with a pharmaceutical composition that comprises ablend of at least a first lipid nanoparticle and a second lipidnanoparticle, wherein the first lipid nanoparticle comprises one or morepolynucleotides. Following the administration or otherwise contactingtargeted cells or tissues with the blended lipid nanoparticlecompositions one or more target cells are transfected with the one ormore polynucleotides encapsulated in one or more of the constituentlipid nanoparticles, such that expression of the one or morepolynucleotides and/or production of one or more functional polypeptidesor proteins is increased or synergistically increased relative to theexpression of the one or more polynucleotides or production of one ormore functional polypeptides or proteins when the first lipidnanoparticle is administered independently of the second lipidnanoparticle. In certain embodiments, the expression of the encapsulatedpolynucleotides that comprise the blended composition may be increased,for example, by at least about two-, four-, five-, ten-, twelve-,fifteen-, twenty-, or twenty-five-, fifty-, seventy-five, one-hundred,two-hundred-, five-hundred-, one thousand-fold, or more relative to theexpression observed when the constituent lipid nanoparticles thatcomprise the blended formulation are independently administered.

Also disclosed herein are pharmaceutical compositions that comprise ablend of a first lipid nanoparticle and a second lipid nanoparticle,wherein the first lipid nanoparticle comprises one or morepolynucleotides. In certain embodiments both the first and the secondlipid nanoparticles comprise or encapsulate the same or a differentpolynucleotide. Upon contacting one or more target cells with thepharmaceutical composition the one or more polynucleotides encapsulatedby the constituent lipid nanoparticles transfect the target cells andare expressed, and where such polynucleotides comprise mRNA, therebyproduce a functional polypeptide or protein. In certain embodiments, theexpression of the one or more polynucleotides by the target cellsexceeds the relative sum of the expression of the one or morepolynucleotides achieved by the first lipid nanoparticle and the secondlipid nanoparticle that comprise the pharmaceutical composition when thetarget cells are contacted with the first lipid nanoparticle and thesecond lipid nanoparticle independently of the other. In otherembodiments, the production of one or more functional polypeptides bytarget cells exceeds the relative sum of the production of one or morefunctional polypeptides achieved by the first lipid nanoparticle and thesecond lipid nanoparticle that comprise the pharmaceutical compositionwhen the target cells are contacted with the first lipid nanoparticleand the second lipid nanoparticle independently of the other.

In certain embodiments, the first lipid nanoparticle or the second lipidnanoparticle comprises one or more cationic lipids. For example, one orboth of the first and second lipid nanoparticles may include one or moreof C12-200, DOTAP (1,2-dioleyl-3-trimethylammonium propane), DODAP(1,2-dioleyl-3-dimethylammonium propane), DOTMA(1,2-di-O-octadecenyl-3-trimethylammonium propane), DLinDMA,DLin-KC2-DMA, HGT4003 and ICE.

In certain embodiments, the first lipid nanoparticle or the second lipidnanoparticle comprise one or more helper lipids. For example, one orboth of the first and second lipid nanoparticles that comprise theblended pharmaceutical compositions may include one or more of helperlipids that are selected from the group consisting of DSPC(1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC(1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE(1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DPPE(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE(1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG(1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)) and cholesterol.

Similarly, in certain embodiments, the first lipid nanoparticle or thesecond lipid nanoparticle may comprise one or more PEG-modified lipids.For example, one or both of the first and second lipid nanoparticles maycomprise one or more of PEG-modified lipids that comprise apoly(ethylene)glycol chain of up to 5 kDa in length covalently attachedto a lipid comprising one or more alkyl chains of C₆-C₂₀ in length.

In certain embodiments, one or more of the at least two lipidnanoparticles that comprise the blended compositions of the inventionare prepared by combining or comprise multiple lipid, non-lipid and/orpolymer components. For example, the first lipid nanoparticle or thesecond lipid nanoparticle may comprise DLinDMA, CHOL, DOPE andDMG-PEG-2000. In some embodiments, the first lipid nanoparticle or thesecond lipid nanoparticle comprises C12-200, CHOL, DOPE andDMG-PEG-2000. In some embodiments, the first lipid nanoparticle or thesecond lipid nanoparticle comprises DLinKC2, CHOL, DOPE andDMG-PEG-2000. Similarly, one or more of the first lipid nanoparticle andthe second lipid nanoparticle may comprise one or more lipids selectedfrom the group consisting of ICE, DSPC, CHOL, DODAP, DOTAP andC8-PEG-2000. In another embodiment, the first lipid nanoparticle or thesecond lipid nanoparticle comprises ICE, DOPE and DMG-PEG-2000. Still inanother embodiment, the first lipid nanoparticle or the second lipidnanoparticle comprises HGT4003, DOPE, CHOL and DMG-PEG-2000.

In certain embodiments, the lipid compositions of the two or moreblended lipid nanoparticles are different (e.g., have different lipidcompositions). For example, contemplated hereby are blended compositionswherein the first lipid nanoparticle comprises a cationic lipid, andwherein the second lipid nanoparticle comprises a cationic lipid whichis different from the cationic lipid which comprises the first lipidnanoparticle

Also disclosed herein are methods of enhancing or otherwise increasing(e.g., synergistically increasing) the delivery or the rate of deliveryof one or more polynucleotides to one or more target cells, as well asmethods of enhancing the residence time of one or more polynucleotideswithin a target cell. Such methods generally comprise contacting the oneor more target cells with a pharmaceutical composition comprising ablend of at least two lipid nanoparticles, each having a different lipidcomposition, such that the delivery of the polynucleotides (e.g., to oneor more target cells, tissues or organs) is enhanced. Upon delivery ofsuch one or more polynucleotides (e.g., one or more antisenseoligonucleotides) to or into the target cells, such polynucleotide mayexert its intended function (e.g., modulate the expression of a targetnucleic acid such as mRNA). For example, an antisense oligonucleotidemay modulate or decrease (e.g., synergistically decrease) the expressionof a targeted gene or nucleic acid. Alternatively, in certainembodiments, following delivery of the polynucleotides to the targetcells the production of a functional peptide or protein encoded by suchpolynucleotide is increased or synergistically increased.

The blended compositions and methods of use described herein may beformulated to specifically target and transfect one or more targetcells, tissues and organs. For example, contemplated target cells maycomprise one or more cells selected from the group consisting ofhepatocytes, epithelial cells, hematopoietic cells, epithelial cells,endothelial cells, lung cells, bone cells, stem cells, mesenchymalcells, neural cells, cardiac cells, adipocytes, vascular smooth musclecells, cardiomyocytes, skeletal muscle cells, beta cells, pituitarycells, synovial lining cells, ovarian cells, testicular cells,fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytesand tumor cells.

In certain embodiments, the blended lipid nanoparticle compositions andmethods of using such blended compositions comprise one or morepolynucleotides (e.g., mRNA). Such polynucleotides may encode, forexample, a functional polypeptide, protein or enzyme, and upon beingexpressed (e.g., translated) by one or more target cells a functionalpolypeptide product (e.g., a protein or enzyme) is produced, and in someinstances secreted by the target cell into the peripheral circulation.In certain embodiments, the one or more of the polynucleotides thatcomprise or are otherwise encapsulated by one or more of the constituentlipid nanoparticles that comprise the blended compositions encode apolypeptide which is aberrantly expressed by the subject. In certainembodiments, the one or more of the encapsulated polynucleotides thatcomprise the blended lipid nanoparticle formulations encode a functionalenzyme such as a urea cycle enzyme (e.g., ornithine transcarbamylase(OTC), carbamoyl-phosphate synthetase 1 (CPS1), argininosuccinatesynthetase (ASS1), argininosuccinate lyase (ASL) or arginase 1 (ARG1)).In certain embodiments the one or more of the encapsulatedpolynucleotides comprises mRNA encoding an enzyme associated with alysosomal storage disorder (e.g., the encapsulated polynucleotide ismRNA encoding one or more of the enzymes agalsidase alfa,alpha-L-iduronidase, iduronate-2-sulfatase,N-acetylglucosamine-1-phosphate transferase, N-acetylglucosaminidase,alpha-glucosaminide acetyltransferase, N-acetylglucosamine 6-sulfatase,N-acetylgalactosamine-4-sulfatase, beta-glucosidase, galactose-6-sulfatesulfatase, beta-galactosidase, beta-glucuronidase, glucocerebrosidase,heparan sulfamidase, hyaluronidase and galactocerebrosidase).Alternatively, in some embodiments, one or more of the encapsulatedpolynucleotides that comprise the blended lipid nanoparticleformulations comprises SEQ ID NO: 2 or SEQ ID NO: 3.

The use of mRNA as the polynucleotide are also contemplated hereby andin particular the use of mRNA that comprises one or more chemicalmodifications. In certain embodiment, such chemical modifications renderthe mRNA more stable and may comprise, for example an end blockingmodification of a 5′ or 3′ untranslated region of the mRNA. In certainembodiments, the chemical modification comprises the inclusion of apartial sequence of a CMV immediate-early 1 (IE1) gene to the 5′untranslated region of the mRNA. In other embodiments the chemicalmodification comprises the inclusion of a poly A tail to the 3′untranslated region of the mRNA. Also contemplated are chemicalmodifications that comprise the inclusion of a Cap1 structure to the 5′untranslated region of the mRNA. In still other embodiments, thechemical modification comprises the inclusion of a sequence encodinghuman growth hormone (hGH) to the 3′ untranslated region of the mRNA.

Also disclosed herein are methods of manipulating the enhanced (e.g.,synergistically increased) expression of the one or more polynucleotidesthat are encapsulated in the one or more of the first or second lipidnanoparticles that comprise the blended compositions and methods ofmanipulating the enhanced production of the polypeptides encoded by suchencapsulated one or more polynucleotides. For example, in certainembodiments the relative ratio of the first and second lipidnanoparticles may be adjusted (e.g., based on the mass of theencapsulated polynucleotide) to enhance expression of one or more of theencapsulated polynucleotides and/or to enhance the production of the oneor more polypeptides encapsulated by such encapsulated polynucleotides.In some embodiments, the ratio of the one or more polynucleotidescomprising the first lipid nanoparticle to the one or morepolynucleotides comprising the second lipid nanoparticle in thepharmaceutical composition is about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1,8:1, 9:1, 10:1, 11:1, 12:1, 15:1, 20:1, 25:1, 30:1, 40:1, 50:1, 60:1,75:1, 100:1, 125:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11,1:12, 1:15, 1:20, 1:25, 1:30, 1:40, 1:50, 1:60, 1:75, 1:100, 1:125 ormore. Similarly, the enhanced (e.g., synergistically increased)expression of the one or more polynucleotides that are encapsulated inthe one or more of the first or second lipid nanoparticles that comprisethe blended compositions may be manipulated by varying the content andrelative concentrations of one or more of the lipids, non-lipids, helperlipids and PEG-modified lipids that comprise such lipid nanoparticles.Furthermore, the enhanced (e.g., synergistically increased) productionby target cells of one or more functional polypeptides or proteinsencoded by encapsulated polynucleotides in the first or second lipidnanoparticles that comprise the blended compositions may be manipulatedby varying the content and relative concentrations of one or more of thelipids, non-lipids, helper lipids and PEG-modified lipids that comprisesuch lipid nanoparticles.

The synergistic enhancements in expression of encapsulatedpolynucleotides that characterize the blended lipid nanoparticleformulations of the present invention and/or the synergistic productionof polypeptides encoded thereby by one or more target cells allowtherapeutically effective concentrations of produced polynucleotides(e.g., a therapeutic protein or enzyme) to be achieved in the targetedtissues (or serum if the polypeptide product is excreted by target cell)using a significantly lower dose of polynucleotide than was previouslyanticipated. Accordingly, in certain embodiments, the effective amountof a polynucleotide required to achieve a desired therapeutic effect maybe reduced by encapsulating such polynucleotide in one or more lipidnanoparticles and blending at least two lipid nanoparticles. Suchmethods comprise a step of administering a pharmaceutical composition tothe subject, wherein the pharmaceutical composition comprises a firstlipid nanoparticle blended with a second lipid nanoparticle, and whereinone or both of the first lipid nanoparticle and the second lipidnanoparticle comprise the polynucleotide, followed by the transfectionof one or more target cells of the subject with such polynucleotides,such that the amount of the polynucleotide required to effectuate atherapeutic effect is reduced (e.g., reduced relative to the amount ofpolynucleotide required to effectuate a therapeutic effect using anon-blended composition or other standard techniques). In certainembodiments, the amount of a polynucleotide required to effectuate atherapeutic effect is reduced by at least about 10%, 15%, 20%, 25%, 30%,40%, 50%, 60%, 75%, 80%, 90%, 95% or 99% relative to the amount ofpolynucleotide required to effectuate a therapeutic effect using anon-blended composition or other standard techniques). In certainembodiments, the amount of a polynucleotide required to effectuate atherapeutic effect is reduced by at least two-, three-, four-, five-,six-, seven-, ten-, twelve-, fifteen-, twenty-, twenty-five-, thirty-,forty- or fifty-fold or more relative to the amount of polynucleotiderequired to effectuate a therapeutic effect using a non-blendedcomposition or other standard techniques).

The above discussed and many other features and attendant advantages ofthe present invention will become better understood by reference to thefollowing detailed description of the invention when taken inconjunction with the accompanying examples. The various embodimentsdescribed herein are complimentary and can be combined or used togetherin a manner understood by the skilled person in view of the teachingscontained herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. is a schematic representing a formulation “blend”, which isgenerally defined as a combination of two or more separate,non-identical formulations into one.

FIG. 2. illustrates the luminescence output of firefly luciferaseprotein in livers of mice following treatment with firefly luciferase(FFL) mRNA-encapsulated lipid nanoparticles based on various cationiclipids. Doses of encapsulated FFL mRNA evaluated were C12-200 (30 μg),DLin-KC2-DMA (90 μg), HGT4003 (200 μg), ICE (200 μg) and DODAP (200 μg).The two controls included a C12-200-based cationic lipid nanoparticleencapsulating a non-fluorescent mRNA (30 μg dose) and PBS vehicle.Values are depicted as median RLU/mg of total protein in liver fourhours post-administration.

FIG. 3. illustrates the Luminescence output of firefly luciferaseprotein in livers of mice four hours after treatment with FFLmRNA-encapsulated lipid nanoparticles. The formulations compared were aC12-200-based formulation (30 μg dose), a DLin-KC2-DMA-based formulation(90 μg dose), a mixed C12-200/DLin-KC2-DMA single formulation (30 μgdose) and a blend of two separate C12-200- and DLin-KC2-DMA-basedformulations. As depicted by FIG. 3, the blended formulation (120 μgdose, 1:3 ratio of C12-200 encapsulated FFL mRNA:DLin-KC2-DMAencapsulated FFL mRNA, respectively) demonstrated a synergisticenhancement of luminescence.

FIG. 4. illustrates a comparison of luminescence output of fireflyluciferase protein in mouse livers based on two ratios of blendedformulations. The individual HGT4003 and ICE formulations were dosed at100 μg of encapsulated firefly luciferase (FFL) mRNA each. The twoblended formulations were administered at a total dose of 200 μgencapsulated FFL mRNA. Values are depicted as median RLU/mg of totalprotein in liver four hours post-administration.

FIG. 5. illustrates the luminescence comparison when blendingformulations encapsulating fluorescent (FFL) and non-fluorescent (GALT)mRNA. The C12-200-based formulations evaluated were dosed at 30 μg,while the DLin-KC2-DMA-based formulations were dosed at 90 μg mRNA. Theblended formulations were dosed at 120 μg total mRNA. Values aredepicted as median RLU/mg of total protein in liver four hourspost-administration.

FIG. 6. illustrates the luminescence comparison of treated livers whenblending formulations encapsulating fluorescent (FFL) with empty lipidnanoparticles (no mRNA). All C12-200-based lipid nanoparticles weredosed at 30 mRNA (or equivalent) while DLin-KC2-DMA-based formulationswere dosed at 90 μg mRNA (or equivalent). Blended formulations weredosed at 120 μg equivalent mRNA. Values are depicted as median RLU/mg oftotal protein in liver four hours post-administration.

FIG. 7. illustrates the luminescence output of firefly luciferaseprotein in the brain tissues of mice following intracerebroventricular(ICV) administration of firefly luciferase (FFL) mRNA-encapsulated lipidnanoparticles. The formulations compared were C12-200-based formulations(0.96 μg dose) and DLin-KC2-DMA-based formulations (2.87 μg dose) bothindividually and relative to a blend of these two formulations in a 1:3ratio (3.83 μg dose). Values are depicted as median RLU/mg of totalprotein in brain four hours post-administration.

FIG. 8. illustrates a comparison of secreted human erythropoietin (EPO)protein following intravenous delivery of EPO mRNA-encapsulated lipidnanoparticles. Blood samples were taken four hours post-injection. Asillustrated in FIG. 8, all formulations demonstrated successful humanEPO secretion by the targeted cells. Each blended formulationdemonstrated a synergistic enhancement of protein production. The dosesare represented in FIG. 8 (in parentheses) as micrograms of encapsulatedhuman EPO mRNA.

FIG. 9. Illustrates synergistic augmentation of protein production whenanalyzed via western blot. A blend of a human EPO mRNA-loadedC12-200-based lipid nanoparticle with an analogous DLin-KC2-DMA-loadedlipid nanoparticle shows a strong band (Lane 4) significant of human EPOprotein isolated from the serum four hours post-administration. The lanecorresponding to an individual C12-200 formulation (Lane 2) yields amoderately detected band while that of the DLin-KC2-DMA-basedformulation (Lane 3) is undetectable.

DETAILED DESCRIPTION

Disclosed herein are pharmaceutical compositions which comprise a blendof at least two lipid nanoparticles, at least one of which comprises apolynucleotide and to related methods of using such pharmaceuticalcompositions as a highly efficient means of increasing the expression ofsuch polynucleotide. Also provided are blended pharmaceuticalcompositions that comprise one or more polynucleotides encoding mRNA andrelated methods of increasing the production of the functionalpolypeptide or protein encoded by such polynucleotide. As used herein,the terms “blend” and “blended” refer to pharmaceutical compositionsthat comprise two or more distinct, heterogeneous lipid nanoparticles.Typically, the two or more distinct, heterogeneous lipid nanoparticlesare combined into a single pharmaceutical composition or formulation.For example, a blended pharmaceutical composition may comprise a firstlipid nanoparticle that comprises the cationic lipid DOTAP and a secondlipid nanoparticle that comprises the cationic lipid ICE. In someembodiments, a blended pharmaceutical composition may comprise a firstlipid nanoparticle that comprises the cationic lipid C12-200 and asecond lipid nanoparticle that comprises the cationic lipid DLinKC2DMA.An illustration of such a blended formulation is depicted in FIG. 1.

It should be noted that the terms “blend” and “blended” as used hereinto describe the pharmaceutical compositions and formulations of thepresent invention are distinguishable from the terms “mix” or “mixture”,which are used herein to describe a pharmaceutical formulation orcomposition that includes only a single lipid nanoparticle in theformulation. For example, the terms mix and mixture are used herein torefer to a pharmaceutical composition comprising only a singlepopulation of lipid nanoparticles, all of which generally have the sameor substantially the same lipid composition. This is in contrast to ablended formulation which comprises two or more different lipidnanoparticles. In particular the terms “mix” and “mixture” are generallyused to describe a pharmaceutical composition or formulation whichincludes a single, homogeneous population of lipid nanoparticles all ofwhich are synthesized from an organic solution containing the samecationic lipids and, for example, any additional helper lipids orPEG-modified lipids.

The blended lipid nanoparticle compositions are characterized as beingable to efficiently deliver the encapsulated polynucleotides to targetcells, and are also characterized by their ability to improve thesubsequent transfection of such encapsulated polynucleotides followingcontacting one or more target cells. The blended lipid nanoparticlecompositions are also characterized by their ability to enhance (e.g.,increase) the expression of polynucleotides encapsulated therein bytarget cells. The blended lipid nanoparticle compositions describedherein are also characterized by their ability to enhance (e.g.,increase) the production of one or more polypeptides or proteins (e.g.,by target cells) encoded by one or more polynucleotides encapsulated insuch nanoparticle compositions. Accordingly, such blended lipidnanoparticle pharmaceutical compositions are also useful for thetreatment of diseases, and in particular the treatment of diseases whichresult from the aberrant expression of genes or gene products (e.g.,diseases associated with the deficient production of a protein). Forexample, the diseases that the blended lipid nanoparticles andpharmaceutical compositions may be used to treat include those in whicha genetic mutation of a particular gene causes the affected cells to notexpress, have reduced expression of, or to express a non-functionalproduct of that gene. Contacting such target cells with the blendedlipid nanoparticles and pharmaceutical compositions such that the targetcells are transfected by the encapsulated polynucleotides increases theexpression of such polynucleotides and increases the production of afunctional protein or polypeptide product that may be useful in thetreatment of disease (e.g., diseases resulting from a protein or enzymedeficiency).

In certain embodiments the blended lipid nanoparticles andpharmaceutical compositions described herein exhibit an increasedability to transfect one or more target cells. As used herein, the terms“transfect” or “transfection” refer to the intracellular introduction ofa polynucleotide into a cell, or preferably into a target cell. Theintroduced polynucleotide may be stably or transiently maintained in thetarget cell. The term “transfection efficiency” refers to the relativeamount of polynucleotide up-taken or introduced by the target cell whichis subject to transfection. In practice, transfection efficiency isestimated by the amount of a reporter polynucleotide product expressedby the target cells following transfection. The blended lipidnanoparticles and pharmaceutical compositions described hereindemonstrate high transfection efficiencies, and in particular suchblends demonstrate high transfection efficiencies relative to thetransfection efficiencies of the individual constituent lipidnanoparticles that comprise such blended compositions. The hightransfection efficiencies observed by the blended lipid nanoparticle andpharmaceutical compositions can minimize adverse effects associated withboth the lipids which comprise the nanoparticles as wells as thepolynucleotides encapsulated by such lipids. Accordingly, the blendedlipid nanoparticles of the present invention demonstrate hightransfection efficacies thereby improving the likelihood thatappropriate dosages of the polynucleotide will be delivered to the siteof pathology, while at the same time minimizing potential systemicadverse effects.

As used herein, the terms “polynucleotide” and “nucleic acid” are usedinterchangeably to refer to genetic material (e.g., DNA or RNA), andwhen such terms are used with respect to the lipid nanoparticlesgenerally refer to the genetic material encapsulated by such lipidnanoparticles. In some embodiments, the polynucleotide is RNA. SuitableRNA includes mRNA, siRNA, miRNA, snRNA and snoRNA. Contemplatedpolynucleotides also include large intergenic non-coding RNA (lincRNA),which generally do not encode proteins, but rather function, forexample, in immune signaling, stem cell biology and the development ofdisease. (See, e.g., Guttman, et al., 458: 223-227 (2009); and Ng, etal., Nature Genetics 42: 1035-1036 (2010), the contents of which areincorporated herein by reference). In certain embodiments, thepolynucleotides of the invention include RNA or stabilized RNA encodinga protein or enzyme (e.g., mRNA encoding human erythropoietin, asrepresented by SEQ ID NO: 3). The present invention contemplates the useof such polynucleotides (and in particular RNA or stabilized RNA) as atherapeutic that is capable of being expressed by target cells tothereby facilitate the production (and in certain instances theexcretion) of a functional enzyme or protein by such target cells, asdisclosed for example, in International Application No.PCT/US2010/058457 and in United States Provisional Application No.PCT/US2012/041724 filed Jun. 8, 2012, the teachings of which are bothincorporated herein by reference in their entirety. For example, incertain embodiments, upon the expression of one or more polynucleotidesby target cells the production of a functional enzyme or protein inwhich a subject is deficient (e.g., a urea cycle enzyme or an enzymeassociated with a lysosomal storage disorder) may be observed. The term“functional”, as used herein to qualify a protein or enzyme, means thatthe protein or enzyme has biological activity, or alternatively is ableto perform the same, or a similar function as the native ornormally-functioning protein or enzyme.

Also provided herein are blended lipid nanoparticles, pharmaceuticalcompositions and related methods for modulating the expression of apolynucleotide and/or for modulating (e.g., increasing) the productionof a functional polypeptide or protein (e.g., an enzyme) encoded by suchpolynucleotide in one or more target cells and tissues. In the contextof the present invention the term “expression” is used in its broadestsense to refer to either the transcription of a specific gene orpolynucleotide into at least one mRNA transcript, or the translation ofat least one mRNA or polynucleotide into a polypeptide (e.g., afunctional protein or enzyme). For example, in certain embodiments theblended lipid nanoparticles comprise at least a first and a second lipidnanoparticle, at least one of which comprise a polynucleotide (e.g.,mRNA) which encodes a functional protein or enzyme. In the context ofpolynucleotides which comprise or encode mRNA, the term expressionrefers to the translation of such mRNA (e.g., by the target cells) toproduce the polypeptide or protein encoded thereby. In the context of anantisense oligonucleotide encapsulated in one or more of the lipidnanoparticle compositions described herein, the term “expression” may beused with reference to one or more targeted genes or nucleic acids(e.g., mRNA). For example, where an encapsulated antisenseoligonucleotide has been prepared to be complementary to a fragment of atarget endogenous mRNA expressed by a cell, the term “expression” may beused with reference to such endogenous mRNA (e.g., the encapsulatedantisense oligonucleotide may reduce the expression of such targetedmRNA).

Blended lipid nanoparticles and pharmaceutical compositions formodulating the expression of aberrantly expressed nucleic acids andpolynucleotides in one or more target cells and tissues are alsoprovided. In certain embodiments such blended lipid nanoparticlescomprise at least a first lipid nanoparticle and a second lipidnanoparticle, one or both of which may encapsulate a polynucleotide. Theblended lipid nanoparticles may comprise, for example, one or morepolynucleotides encapsulated in a first lipid nanoparticle which isblended with one or more different lipid nanoparticles (i.e., a secondor third lipid nanoparticle) that can optionally encapsulate one or morepolynucleotides. Such blended lipid nanoparticle formulationsdemonstrate enhanced expression of the one or more polynucleotidesencapsulated thereby relative to the expression of the samepolynucleotides observed following the administration of a single lipidnanoparticle (e.g., the first lipid nanoparticle). Similarly, suchblended lipid nanoparticle formulations may demonstrate enhancedproduction of one or more polypeptides (e.g., a functional enzyme)encoded by an encapsulated polynucleotides relative to the production ofthe same polypeptides observed following the administration of a singlelipid nanoparticle (e.g., the first lipid nanoparticle). For example, incertain embodiments the blended lipid nanoparticles and pharmaceuticalcompositions are capable of increasing the expression of encapsulatedpolynucleotides, and/or the production of the polypeptides encoded bysuch encapsulated polynucleotides, in a target cell by at least aboutten-fold, twenty-fold, thirty-fold, forty-fold, fifty-fold, sixty-fold,seventy-fold, one-hundred-fold, five-hundred-fold, one-thousand-fold, ormore relative to the expression of polynucleotide or production ofpolypeptide observed when the target cell is contacted with suchpolynucleotides encapsulated in a single lipid nanoparticle.

Methods of enhancing (e.g., increasing) the expression of apolynucleotide and methods of increasing the production and secretion ofa functional polypeptide product in target cells and tissues (e.g.,hepatocytes) are also provided. In some embodiments, the targeted cellsor tissues aberrantly express the polynucleotide encapsulated by one ormore of the lipid nanoparticles that comprise the blended pharmaceuticalcomposition. Also provided herein are methods of increasing theexpression of one or more polynucleotides (e.g., mRNA) and methods ofincreasing the production and/or secretion of one or more polypeptides(e.g., a functional enzyme) in one or more target cells, tissues andorgans. Generally, such methods comprise contacting the target cellswith a pharmaceutical composition that comprises a blend of at least twolipid-based nanoparticles (e.g., a first and a second lipidnanoparticle), wherein at least one of such lipid-based nanoparticlescomprises or otherwise encapsulates the one or more polynucleotides.

Also provided herein are methods and compositions for enhancing (e.g.,increasing) or otherwise modulating the expression of one or morepolynucleotides (e.g., mRNA) and/or enhancing (e.g., increasing) orotherwise modulating the production and secretion of the polypeptides orproteins encoded thereby in the targeted cells of a subject. Suchmethods generally comprise the step of administering (e.g.,intravenously administering) a blended pharmaceutical composition to asubject wherein such pharmaceutical composition comprise at least twoblended lipid nanoparticles (e.g., a first and a second lipidnanoparticle), at least one of which encapsulates or otherwise comprisesone or more polynucleotides. In certain embodiments, the step ofadministering the blended pharmaceutical composition to a subjectfacilitates the contacting of the blended lipid nanoparticles with thetargeted cells and tissues, the result of which is an enhanced (e.g.,increased) expression of the encapsulated polynucleotides and enhanced(e.g., increased) product of the polypeptide encoded by suchencapsulated polynucleotides by the contacted target cells and tissues.As the term “enhanced” is used herein to describe the activity ofblended lipid nanoparticles, it should be noted that such enhancement isgenerally determined relative to the sum of the effect observed orproduced by the constituent members that comprise the blended lipidnanoparticle formulation. For example, the enhanced expression of apolynucleotide and/or the enhanced production or secretion of apolypeptide which is observed following the administration of a blendedlipid nanoparticle formulation comprising two different lipidnanoparticles designated “A” and “B”, wherein only lipid nanoparticle“A” encapsulates the polynucleotide, is enhanced relative to the sum ofthe expression of polynucleotide and/or production or secretion ofpolypeptide observed by both “A” and “B” when administered independentlyof one another.

The present inventors have discovered that in certain embodiments theexpression of the polynucleotides (and the corresponding productionand/or secretion of a polypeptide encoded by polynucleotides comprisingmRNA) observed when such polynucleotides are administered in a blendedlipid-based nanoparticle composition is enhanced and in many instancesexceeds (e.g., by about two-, three-, four-, five-, ten-, twenty-,twenty-five-, thirty-, forty-, fifty-, sixty-, seventy-eighty, ninety,one-hundred, two-hundred-fold or more) the expression (and thecorresponding production and/or secretion of a polypeptide where suchpolynucleotides comprise mRNA) observed when such polynucleotides areadministered using each of the two or more independent constituent lipidnanoparticles which comprise the blended lipid nanoparticlepharmaceutical compositions. Accordingly, the blended pharmaceuticalcompositions (e.g., a single pharmaceutical composition comprising twoseparate, non-identical lipid nanoparticles) demonstrate a synergistic(i.e., non-additive) increase in the expression of the polynucleotidesencapsulated in the two or more lipid nanoparticles which comprise thepharmaceutical composition and a synergistic (i.e., non-additive)increase in the production and/or secretion of the polypeptides encodedby such encapsulated polynucleotides where such polynucleotides comprisemRNA. The synergistic increase is evident relative to the additive totalexpression of such polynucleotides (or the additive total production ofpolypeptides encoded by polynucleotides comprising mRNA), that isobserved when each of the constituent lipid nanoparticles which comprisethe blended pharmaceutical composition are administered individually.The observed synergistic increases in the expression of encapsulatedpolynucleotides (and/or production of polypeptide encoded bypolynucleotides comprising mRNA) is evident across a variety of lipidnanoparticles and at various ratios of such blended lipid nanoparticles.Furthermore, the observed enhanced expression of polynucleotidesencapsulated in at least one of the lipid nanoparticles which comprisethe blended pharmaceutical compositions (and the correspondingproduction of the polypeptides encoded by encapsulated polynucleotidescomprising mRNA) may range from about 1.5- to 25-fold increases, or moreas compared to the sum of the expression (or where applicable theproduction and/or secretion) observed in the constituent lipidnanoparticles which comprise such blended pharmaceutical compositions,thereby demonstrating that blending of two or more separate lipidnanoparticles (at least one of which encapsulates a polynucleotide)mechanistically allows the greater expression of such polynucleotides,which in turn corresponds to a greater production of the product encodedthereby, as compared to the separate lipid nanoparticles comprising suchblended pharmaceutical composition.

The mechanism of this synergistic augmentation in protein production hasnot yet been elucidated. Without wishing to be bound by any oneparticular theory, possible explanations include, for example,non-competing pathways or mechanisms of cellular entry by each of thelipid nanoparticle formulations that comprise the blended pharmaceuticalcomposition, the combination of different intracellular traffickingmechanisms (e.g., proton-sponge vs. fusogenicity), the combination ofdrug release properties with endosomal release properties and/or themodulation of active inhibitory pathways allowing greater uptake ofnanoparticles.

The blended pharmaceutical compositions, and in particular theconstituent lipid nanoparticles which comprise such blendedcompositions, are capable of delivering polynucleotides of varying sizesto their target cells or tissues. In certain embodiments, the lipidnanoparticles of the present invention are capable of delivering largepolynucleotide sequences (e.g., polynucleotides of at least 1 kb, 1.5kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9kb, 10 kb, 12 kb, 15 kb, 20 kb, 25 kb, 30 kb, or more).

In certain embodiments, the blended pharmaceutical compositions and theencapsulated polynucleotides comprised therein may be formulated withone or more acceptable excipients or reagents to facilitate the deliveryof such polynucleotides to the intended or targeted cells, tissues andorgans. Appropriate reagents and excipients may be generally selectedwith regards to a number of factors, which include, among other things,the biological or chemical properties of the polynucleotides (e.g., thecharge), the intended route of administration, the anticipatedbiological environment to which such polynucleotides will be exposed andthe specific properties of the intended target cells and tissues. Insome embodiments, one or more of the lipid nanoparticles which comprisethe blended pharmaceutical composition demonstrate a preferential and/orsubstantial binding to a target cell relative to non-target cells. Insome embodiments, the lipid nanoparticles which comprise the blendedpharmaceutical formulation, and in particular the lipid nanoparticleswhich encapsulate one or more polynucleotides, deliver their contents tothe target cell such that the polynucleotides are delivered to theappropriate subcellular compartment, such as the cytoplasm, and may beexpressed accordingly, such that, in certain embodiments one or morefunctional polypeptides (e.g., a functional protein) is produced and/orexcreted by the target cell.

In certain embodiments, the blended pharmaceutical compositions comprisetwo or more separate lipid nanoparticles (e.g., a first lipidnanoparticle comprising HGT4003, DOPE, cholesterol and DMG-PEG2K blendedwith a second lipid nanoparticle comprising ICE, DOPE and DMG-PEG2K).The first lipid nanoparticle and the second lipid nanoparticle whichcomprise the blended pharmaceutical composition may each encapsulate thesame one or more polynucleotides. A synergistic increase in theexpression of the one or more polynucleotides by the target cellsfollowing the administration of the blended pharmaceutical compositionto a subject exceeds the relative sum of the expression of the one ormore polynucleotides achieved by the first lipid nanoparticle and theexpression of the one or more polynucleotides achieved by the secondlipid nanoparticle when the first lipid nanoparticle and the secondlipid nanoparticle are administered to the subject independently.Similarly, in certain embodiments a synergistic increase in theproduction one or more functional polypeptides encoded by one or moreencapsulated polynucleotides by the target cells followingadministration of the blended pharmaceutical composition to a subjectexceeds the relative sum of the production of the one or more functionalpolypeptides achieved by the first lipid nanoparticle and the productionof the one or more polypeptides achieved by the second lipidnanoparticle when the first lipid nanoparticle and the second lipidnanoparticle are administered to the subject independently.Alternatively, in certain embodiments only one of the lipidnanoparticles (e.g., the first lipid nanoparticles) which comprise theblended pharmaceutical composition encapsulates one or morepolynucleotides.

In some embodiments, the blended lipid nanoparticles and blendedpharmaceutical compositions are capable of enhancing the expression ofone or more polynucleotides irrespective whether some or all of thelipid nanoparticles which comprise the blended pharmaceuticalcomposition encapsulate one or more polynucleotide. In particular, theblended lipid nanoparticles which comprise a first lipid nanoparticleencapsulating a polynucleotide and a second empty lipid nanoparticle(i.e., not encapsulating a polynucleotide) are capable ofsynergistically enhancing the expression of such polynucleotides (e.g.,increasing expression about two-, four-, five-, ten-, twenty-,twenty-five-, thirty-, forty-, fifty-, one-hundred-, two-hundred-,five-hundred-, one thousand-fold or more). Accordingly, in the contextof the present invention, at least one of the liposomal lipidnanoparticle components of the blended formulation serves to transportthe polynucleotide to the target cell. In some embodiments two of the atleast two lipid nanoparticle components of the blended formulation serveto transport one or more polynucleotides to the target cell. Uponcontacting the targeted cells, such blended pharmaceutical compositionsdemonstrate an increase in the expression of the encapsulatedpolynucleotide (e.g., by at least about two-, five-, ten- ortwenty-fold) and, in certain embodiments thereby increases theproduction of a functional polypeptide encoded by such encapsulatedpolynucleotide.

As used herein, the phrase “lipid nanoparticle” refers to a vesicle orcarrier comprising one or more lipids (e.g., cationic and/ornon-cationic lipids). Examples of suitable lipids include, for example,the cationic lipids such as C12-200, ICE, DLin-KC2-DMA, DOPE,DMG-PEG-2000, HGT4003, non-cationic lipids, cholesterol-based lipids,helper lipids, PEG-modified lipids, as well as the phosphatidylcompounds (e.g., phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides) and combinations or mixtures of theforgoing. The blended pharmaceutical compositions described hereincomprise at least two or more of the lipid nanoparticles each of whichhave a different lipid composition. Such two or more lipid nanoparticleswhich comprise the blended compositions are referred to herein as a“first lipid nanoparticle”, “second lipid nanoparticle”, “third lipidnanoparticle” and so forth. The designations of, for example first andsecond lipid nanoparticles are made for the purpose of distinguishingthe different lipid nanoparticles that comprise the blendedpharmaceutical compositions and are not intended to limit the number ofdifferent lipid nanoparticles that comprise such blended pharmaceuticalcompositions.

The present inventions also contemplate the use blended pharmaceuticalcompositions comprising one or more lipid nanoparticles that compriseone or more cationic lipids. As used herein, the phrase “cationic lipid”refers to any of a number of lipid species that carry a net positivecharge at a selected pH, such as physiological pH. The contemplatedlipid nanoparticles may be prepared by including multi-component lipidmixtures of varying ratios employing one or more cationic lipids,non-cationic lipids and PEG-modified lipids. In certain embodiments,each of the first and second lipid nanoparticles that comprise theblended pharmaceutical formulations comprise one or more cationiclipids. Similarly, also contemplated are blended pharmaceuticalcompositions that comprise two or more separate lipid nanoparticles,wherein such lipid nanoparticles comprise cationic lipids each havingdifferent lipid compositions. For example, in certain embodiments, thefirst and second lipid nanoparticles each comprise a different cationiclipid (e.g., the first lipid nanoparticle comprises ICE and the secondlipid nanoparticle comprises DLin-KC2-DMA).

Several cationic lipids have been described in the literature, many ofwhich are commercially available. Cationic lipids may include, but arenot limited to ALNY-100((3aR,5s,6aS)-N,N-dimethyl-2,2-dn(9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine)), DODAP (1,2-dioleyl-3-dimethylammonium propane),HGT4003 (WO 2012/170889, the teachings of which are incorporated hereinby reference in their entirety), HGT5000 (U.S. Provisional PatentApplication No. 61/617,468, the teachings of which are incorporatedherein by reference in their entirety) or HGT5001(cis or trans)(Provisional Patent Application No. 61/617,468), aminoalcohol lipidoidssuch as those disclosed in WO2010/053572, DOTAP(1,2-dioleyl-3-trimethylammonium propane), DOTMA(1,2-di-O-octadecenyl-3-trimethylammonium propane), DLinDMA(1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane)(Heyes, et al., J. Contr.Rel. 107:276-287(2005)), DLin-KC2-DMA (Semple, et al., Nature Biotech.28:172-176 (2010)), C12-200 (Love, et al., Proc. Nat'l. Acad. Sci.107:1864-1869(2010)),N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride. (Feigneret al. (Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S. Pat. No.4,897,355). DOTMA can be formulated alone or can be combined withdioleoylphosphatidylethanolamine or “DOPE” or other cationic ornon-cationic lipids into a lipid nanoparticle. Other suitable cationiclipids include, for example, 5-carboxyspermylglycinedioctadecylamide or“DOGS,”2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminiumor “DOSPA” (Behr et al. Proc. Nat.'l Acad. Sci. 86, 6982 (1989); U.S.Pat. Nos. 5,171,678; 5,334,761), 1,2-Dioleoyl-3-Dimethylammonium-Propaneor “DODAP”, 1,2-Dioleoyl-3-Trimethylammonium-Propane or “DOTAP”.Contemplated cationic lipids also include1,2-distearyloxy-N,N-dimethyl-3-aminopropane or “DSDMA”,1,2-dioleyloxy-N,N-dimethyl-3-aminopropane or “DODMA”,1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or “DLinDMA”,1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane or “DLenDMA”,N-dioleyl-N,N-dimethylammonium chloride or “DODAC”,N,N-distearyl-N,N-dimethylammonium bromide or “DDAB”,N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide or “DMRIE”,3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propaneor “CLinDMA”, 2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethy1-1-(cis,cis-9′, 1-2′-octadecadienoxy)propane or “CpLinDMA”,N,N-dimethyl-3,4-dioleyloxybenzylamine or “DMOBA”,1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane or “DOcarbDAP”,2,3-Dilinoleoyloxy-N,N-dimethylpropylamine or “DLinDAP”,1,2-N,N′-Dilinoleylcarbamyl-3-dimethylaminopropane or “DLincarbDAP”,1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane or “DLinCDAP”,2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane or “DLin-K-DMA”,2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane or“DLin-K-XTC2-DMA”, or mixtures thereof. (Heyes, J., et al., J ControlledRelease 107: 276-287 (2005); Morrissey, D V., et al., Nat. Biotechnol.23(8): 1003-1007 (2005); PCT Publication WO2005/121348A1).

The use of cholesterol-based cationic lipids to formulate the blendedlipid nanoparticles is also contemplated by the present invention. Suchcholesterol-based cationic lipids can be used, either alone or incombination with other cationic or non-cationic lipids. Suitablecholesterol-based cationic lipids include, for example, DC-Chol(N,N-dimethyl-N-ethylcarboxamidocholesterol),1,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem. Biophys.Res. Comm 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997);U.S. Pat. No. 5,744,335).

In addition, several reagents are commercially available to enhancetransfection efficacy. Suitable examples include LIPOFECTIN (DOTMA:DOPE)(Invitrogen, Carlsbad, Calif.), LIPOFECTAMINE (DOSPA:DOPE) (Invitrogen),LIPOFECTAMINE2000. (Invitrogen), FUGENE, TRANSFECTAM (DOGS), andEFFECTENE.

Also contemplated are cationic lipids such as the dialkylamino-based,imidazole-based, and guanidinium-based lipids. For example, certainembodiments are directed to a composition comprising one or moreimidazole-based cationic lipids, for example, the imidazole cholesterolester or “ICE” lipid (3S, 10R, 13R, 17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl3-(1H-imidazol-4-yl)propanoate, as represented by structure (I) below.In certain embodiments, a lipid nanoparticle for delivery of RNA (e.g.,mRNA) encoding a functional protein may comprise one or moreimidazole-based cationic lipids, for example, the imidazole cholesterolester or “ICE” lipid (3S, 10R, 13R, 17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl3-(1H-imidazol-4-yl)propanoate, as represented by structure (I).

Without wishing to be bound by a particular theory, it is believed thatthe fusogenicity of the imidazole-based cationic lipid ICE is related tothe endosomal disruption which is facilitated by the imidazole group,which has a lower pKa relative to traditional cationic lipids. Theendosomal disruption in turn promotes osmotic swelling and thedisruption of the liposomal membrane, followed by the transfection orintracellular release of the polynucleotide contents loaded orencapsultated therein into the target cell.

The imidazole-based cationic lipids are also characterized by theirreduced toxicity relative to other cationic lipids. In some embodiments,one or more of the lipid nanoparticles which comprises the blendedpharmaceutical composition comprise an imidazole-based cationic lipidsuch as ICE, to reduce the relative concentration of other more toxiccationic lipids in such blended pharmaceutical composition. Theimidazole-based cationic lipids (e.g., ICE) may be used as the solecationic lipid in one or more of the lipid nanoparticles that comprisethe blended formulations, or alternatively may be combined withtraditional cationic lipids (e.g., DOPE, DC-Chol), non-cationic lipids,PEG-modified lipids and/or helper lipids. The cationic lipid maycomprise a molar ratio of about 1% to about 90%, about 2% to about 70%,about 5% to about 50%, about 10% to about 40% of the total lipid presentin the lipid nanoparticle, or preferably about 20% to about 70% of thetotal lipid present in the lipid nanoparticle.

Similarly, certain embodiments are directed to lipid nanoparticlescomprising the HGT4003 cationic lipid2-((2,3-Bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)propyl)disulfanyl)-N,N-dimethylethanamine,as represented by structure (II) below, and as further described in U.S.Provisional Application No: PCT/US2012/041663, filed Jun. 8, 2012, theentire teachings of which are incorporated herein by reference in theirentirety:

In other embodiments the compositions and methods described herein aredirected to lipid nanoparticles comprising one or more cleavable lipids,such as, for example, one or more cationic lipids or compounds thatcomprise a cleavable disulfide (S—S) functional group (e.g., HGT4001,HGT4002, HGT4003, HGT4004 and HGT4005), as further described in U.S.Provisional Application No: PCT/US2012/041663.

The use and inclusion of polyethylene glycol (PEG)-modifiedphospholipids and derivatized lipids such as derivatized cerarmides(PEG-CER), including N-Octanoyl-Sphingosine-1-[Succinyl(MethoxyPolyethylene Glycol)-2000] (C8 PEG-2000 ceramide) is also contemplatedby the blended lipid nanoparticle formulations of the present invention,either alone or preferably in combination with other lipid formulationswhich comprise one or more of the lipid nanoparticles which comprise ablended pharmaceutical composition. Contemplated PEG-modified lipidsinclude, but are not limited to, a polyethylene glycol chain of up to 5kDa in length covalently attached to a lipid with alkyl chain(s) ofC₆-C₂₀ length. The addition of such components may prevent complexaggregation and may also provide a means for increasing circulationlifetime and increasing the delivery of the lipid-polynucleotidecomposition to the target tissues, (Klibanov et al. (1990) FEBS Letters,268 (1): 235-237), or they may be selected to rapidly exchange out ofthe formulation in vivo (see U.S. Pat. No. 5,885,613). Particularlyuseful exchangeable lipids are PEG-ceramides having shorter acyl chains(e.g., C14 or C18). The PEG-modified phospholipid and derivitized lipidsof the present invention may comprise a molar ratio from about 0% toabout 20%, about 0.5% to about 20%, about 1% to about 15%, about 4% toabout 10%, or about 2% of the total lipid present in a liposomal lipidnanoparticle.

While lipid-based vehicles and their component lipids present promisingmeans of delivering their polynucleotide contents intracellularly, theutility of many lipids (and in particular cationic lipids) may belimited by their associated cytotoxicity. This is particularly true ofLIPOFECTIN, the DOTMA component of which is not-readily degraded in vivoand is toxic to cells and tissues. The blended lipid nanoparticlecompositions of the present invention provide means of reducing thetoxicities associated with lipids, and in particular the toxicityassociated with cationic lipids. In certain embodiments, the synergisticenhancement in expression of encapsulated polynucleotides observed withthe use of the blended lipid nanoparticle compositions of the presentinvention may correspond to reduced amounts of lipids (and in particulartoxic lipids) necessary to effectuate the transfection of an effectiveamount of such polynucleotides to one or more target cells. Accordingly,also contemplated herein are methods for mediating, reducing oreliminating the toxicity associated with one or more lipids, and inparticular one or more cationic lipids. For example, the amount ofcationic lipid required to effectuate the transfection of an effectiveamount of one or more polynucleotides into one or more target cells maybe reduce by incorporating such cationic lipid into a first lipidnanoparticle composition and blending such first lipid nanoparticlecomposition with a second lipid nanoparticle. The enhanced expression ofthe polynucleotide observed with the use of such blended lipidnanoparticle composition may permit a corresponding reduction in theamount of such lipid required to effectuate the transfection of aneffective amount of such one or more polynucleotides into such one ormore target cells. In certain amounts, the toxicity of the one or morelipids is reduced by at least about 10%, 15%, 20%, 25%, 30%, 40%, 50%,60%, 75%, 80%, 90%, 95%, 99% or alternatively is eliminated.

The present invention also contemplates the use of non-cationic lipidsin one or more of the lipid nanoparticles which comprise the blendedformulations. As used herein, the phrase “non-cationic lipid” refers toany neutral, zwitterionic or anionic lipid. As used herein, the phrase“anionic lipid” refers to any of a number of lipid species that carry anet negative charge at a selected pH, such as physiological pH.Non-cationic lipids include, but are not limited to,distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof. Such non-cationic lipids may be used alone, but arepreferably used in combination with other excipients, for example,cationic lipids. When used in combination with a cationic lipid, thenon-cationic lipid may comprise a molar ratio of 5% to about 90%, orpreferably about 10% to about 70% of the total lipid present in thelipid nanoparticle.

Also contemplated is inclusion of polymers in the lipid nanoparticlesthat comprise the blended pharmaceutical formulation. Suitable polymersmay include, for example, polyacrylates, polyalkycyanoacrylates,polylactide, polylactide-polyglycolide copolymers, polycaprolactones,dextran, albumin, gelatin, alginate, collagen, chitosan, cyclodextrinsand polyethylenimine.

In certain embodiments, the lipid nanoparticles are formulated based inpart upon their ability to facilitate the transfection of apolynucleotide to a target cell. In another embodiment, the lipidnanoparticles may be selected and/or prepared to optimize delivery ofpolynucleotides to a target cell, tissue or organ. For example, if thetarget cell is a hepatocyte the properties of the lipid nanoparticle(e.g., size, charge and/or pH) may be optimized to effectively deliversuch lipid nanoparticle to the target cell or organ, reduce immuneclearance and/or promote retention in that target organ. Alternatively,if the target tissue is the central nervous system the selection andpreparation of the lipid nanoparticle must consider penetration of, andretention within the blood brain barrier and/or the use of alternatemeans of directly delivering such lipid nanoparticles to such targettissue (e.g., intracerebrovascular administration). In certainembodiments, the blended compositions or its constituent lipidnanoparticles may be combined with agents that facilitate the transferof encapsulated polynucleotides (e.g., agents which disrupt or improvethe permeability of the blood brain barrier and thereby enhance thetransfer of such encapsulated polynucleotides to the target cells).While lipid nanoparticles can facilitate introduction of polynucleotidesinto target cells, the addition of polycations (e.g., poly L-lysine andprotamine) to the lipid nanoparticles or the blended pharmaceuticalcompositions as a copolymer can also facilitate, and in some instancesmarkedly enhance the transfection efficiency of several types ofcationic liposomes by 2-28 fold in a number of cell lines both in vitroand in vivo. (See N. J. Caplen, et al., Gene Ther. 1995; 2: 603; S. Li,et al., Gene Ther. 1997; 4, 891.)

For the purposes of the present invention, at least one of the lipidnanoparticles (e.g., the first lipid nanoparticle) that comprise theblended pharmaceutical composition is prepared to encapsulate thedesired one or more polynucleotides. In some embodiments all of thelipid nanoparticles that comprise a blended pharmaceutical compositionare prepared to encapsulate one or more polynucleotides. For example,the blended first and second lipid nanoparticles that comprise theblended composition may each encapsulate the same polynucleotides (e.g.,mRNA encoding a deficient enzyme), or alternatively may each encapsulatea different polynucleotide. The process of incorporating a desiredpolynucleotide (e.g., mRNA) into a liposome or a lipid nanoparticle isreferred to herein as or “loading” or “encapsulating” (Lasic, et al.,FEBS Lett., 312: 255-258, 1992). The lipid nanoparticle-loaded or-encapsulated polynucleotides may be completely or partially located inthe interior space of the lipid nanoparticle, within the bilayermembrane of the lipid nanoparticle, or associated with the exteriorsurface of the lipid nanoparticle.

Loading or encapsulating a polynucleotide into a lipid nanoparticle mayserve to protect the polynucleotide from an environment which maycontain enzymes or chemicals (e.g., serum) that degrade polynucleotidesand/or systems or receptors that cause the rapid excretion of thepolynucleotides. Accordingly, in some embodiments, the selected lipidnanoparticles that comprise the blended pharmaceutical compositions arecapable of enhancing the stability of the polynucleotide(s) encapsulatedthereby, particularly with respect to the environments into which suchpolynucleotides will be exposed. Encapsulating the polynucleotides intoone or more of the lipid nanoparticles which comprise the blendedpharmaceutical compositions also facilitates the delivery of suchpolynucleotides into the target cells and tissues. For example, thelipid nanoparticles can allow the encapsulated polynucleotide to reachthe target cell or may preferentially allow the encapsulatedpolynucleotide to reach the target cells or organs on a discriminatorybasis (e.g., the lipid nanoparticles may concentrate in the liver orspleens of a subject to which the blended composition is administered).Alternatively, the lipid nanoparticles may limit the delivery ofencapsulated polynucleotides to other non-targeted cells or organs wherethe presence of the encapsulated polynucleotides may be undesirable orof limited utility.

Preferably, the lipid nanoparticles are prepared by combining multiplelipid and/or polymer components. For example, a lipid nanoparticle maybe prepared using DSPC/CHOL/DODAP/C8-PEG-5000 ceramide in a molar ratioof about 1 to 50: 5 to 65: 5 to 90: 1 to 25, respectively. A lipidnanoparticle may be comprised of additional lipid combinations invarious ratios, including for example, DSPC/CHOL/DODAP/mPEG-5000 (e.g.,combined at a molar ratio of about 33:40:25:2), DSPC/CHOL/DODAP/C8PEG-2000-Cer (e.g., combined at a molar ratio of about 31:40:25:4),POPC/DODAP/C8-PEG-2000-Cer (e.g., combined at a molar ratio of about75-87:3-14:10) or DSPC/CHOL/DOTAP/C8 PEG-2000-Cer (e.g., combined at amolar ratio of about 31:40:25:4). The selection of cationic lipids,non-cationic lipids and/or PEG-modified lipids which comprise the lipidnanoparticles, as well as the relative molar ratio of such lipids toeach other, is based upon the characteristics of the selected lipid(s),the nature of the intended target cells or tissues and thecharacteristics of the polynucleotides to be delivered by the lipidnanoparticle. Additional considerations include, for example, thesaturation of the alkyl chain, as well as the size, charge, pH, pKa,fusogenicity and toxicity of the selected lipid(s).

The lipid nanoparticles for use in the present invention can be preparedby various techniques which are presently known in the art.Multi-lamellar vesicles (MLV) may be prepared conventional techniques,for example, by depositing a selected lipid on the inside wall of asuitable container or vessel by dissolving the lipid in an appropriatesolvent, and then evaporating the solvent to leave a thin film on theinside of the vessel or by spray drying. An aqueous phase may then addedto the vessel with a vortexing motion which results in the formation ofMLVs. Uni-lamellar vesicles (ULV) can then be formed by homogenization,sonication or extrusion of the multi-lamellar vesicles. In addition,unilamellar vesicles can be formed by detergent removal techniques.

In certain embodiments, the blended compositions of the presentinvention comprise a lipid nanoparticle wherein the polynucleotide(e.g., mRNA encoding OTC) is associated on both the surface of the lipidnanoparticle (e.g., a liposome) and encapsulated within the same lipidnanoparticle. For example, during preparation of the compositions of thepresent invention, cationic lipids which comprise the lipidnanoparticles may associate with the polynucleotides (e.g., mRNA)through electrostatic interactions with such therapeutic mRNA.

In certain embodiments, the blended compositions of the presentinvention may be loaded with diagnostic radionuclide, fluorescentmaterials or other materials that are detectable in both in vitro and invivo applications. For example, suitable diagnostic materials for use inthe present invention may includeRhodamine-dioleoylphosphatidylethanolamine (Rh-PE), Green FluorescentProtein mRNA (GFP mRNA), Renilla Luciferase mRNA and Firefly LuciferasemRNA.

During the preparation of liposomal lipid nanoparticles, water solublecarrier agents may be also encapsulated in the aqueous interior byincluding them in the hydrating solution, and lipophilic molecules maybe incorporated into the lipid bilayer by inclusion in the lipidformulation. In the case of certain molecules (e.g., cationic or anioniclipophilic polynucleotides), loading of the polynucleotide intopreformed lipid nanoparticles or liposomes may be accomplished, forexample, by the methods described in U.S. Pat. No. 4,946,683, thedisclosure of which is incorporated herein by reference. Followingencapsulation of the polynucleotide, the lipid nanoparticles may beprocessed to remove un-encapsulated mRNA through processes such as gelchromatography, diafiltration or ultrafiltration. For example, if it isdesirous to remove externally bound polynucleotide from the surface ofthe lipid nanoparticles which comprise the blended pharmaceuticalcompositions, such lipid nanoparticles may be subject to aDiethylaminoethyl SEPHACEL column.

In addition to the encapsulated polynucleotide, one or more therapeuticor diagnostic agents may be included or encapsulated in the lipidnanoparticle. For example, such additional therapeutic agents may beassociated with the surface of the lipid nanoparticle, can beincorporated into the lipid bilayer of the lipid nanoparticle byinclusion in the lipid formulation or loading into preformed lipidnanoparticles (see U.S. Pat. Nos. 5,194,654 and 5,223,263, which areincorporated by reference herein).

There are several methods for reducing the size, or “sizing”, of lipidnanoparticles, and any of these methods may generally be employed whensizing is used as part of the invention. The extrusion method is a onemethod of liposome sizing. (Hope, M J et al. Reduction of Liposome Sizeand Preparation of Unilamellar Vesicles by Extrusion Techniques. In:Liposome Technology (G. Gregoriadis, Ed.) Vol. 1. p 123 (1993). Themethod consists of extruding liposomes through a small-porepolycarbonate membrane or an asymmetric ceramic membrane to reduceliposome sizes to a relatively well-defined size distribution.Typically, the suspension is cycled through the membrane one or moretimes until the desired liposome size distribution is achieved. Theliposomes may be extruded through successively smaller pore membranes toachieve gradual reduction in liposome size.

A variety of alternative methods known in the art are available forsizing of a population of lipid nanoparticles. One such sizing method isdescribed in U.S. Pat. No. 4,737,323, incorporated herein by reference.Sonicating a liposome or lipid nanoparticle suspension either by bath orprobe sonication produces a progressive size reduction down to small ULVless than about 0.05 microns in diameter. Homogenization is anothermethod that relies on shearing energy to fragment large liposomes intosmaller ones. In a typical homogenization procedure, MLV arerecirculated through a standard emulsion homogenizer until selectedliposome sizes, typically between about 0.1 and 0.5 microns, areobserved. The size of the lipid nanoparticles may be determined byquasi-electric light scattering (QELS) as described in Bloomfield, Ann.Rev. Biophys. Bioeng., 10:421-450 (1981), incorporated herein byreference. Average lipid nanoparticle diameter may be reduced bysonication of formed lipid nanoparticles. Intermittent sonication cyclesmay be alternated with QELS assessment to guide efficient liposomesynthesis.

Selection of the appropriate size of a lipid nanoparticle must take intoconsideration the site of the target cell or tissue and to some extentthe application for which the lipid nanoparticle is being made. As usedherein, the phrase “target cell” refers to cells to which one or more ofthe lipid nanoparticles which comprise the blended composition are to bedirected or targeted. In some embodiments, the target cells comprise aparticular tissue or organ. In some embodiments, the target cells aredeficient in a protein or enzyme of interest. For example, where it isdesired to deliver a polynucleotide to a hepatocyte, the hepatocyterepresents the target cell. In some embodiments, the polynucleotides andblended compositions of the present invention transfect the target cellson a discriminatory basis (i.e., do not transfect non-target cells). Thecompositions and methods of the present invention may be prepared topreferentially target a variety of target cells, which include, but arenot limited to, hepatocytes, epithelial cells, hematopoietic cells,epithelial cells, endothelial cells, lung cells, bone cells, stem cells,mesenchymal cells, neural cells (e.g., meninges, astrocytes, motorneurons, cells of the dorsal root ganglia and anterior horn motorneurons), photoreceptor cells (e.g., rods and cones), retinal pigmentedepithelial cells, secretory cells, cardiac cells, adipocytes, vascularsmooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells,pituitary cells, synovial lining cells, ovarian cells, testicular cells,fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytesand tumor cells.

Following transfection of one or more target cells by thepolynucleotides encapsulated in the one or more lipid nanoparticlescomprising the blended composition, the production of the product (e.g.,a functional polypeptide or protein) encoded by such polynucleotide maybe preferably stimulated and the capability of such target cells toexpress the polynucleotide and produce, for example, a polypeptide orprotein of interest is enhanced. For example, transfection of a targetcell by the blended compositions encapsulating OTC mRNA will enhance(i.e., increase) the expression of the OTC mRNA and produce a functionalOTC enzyme.

In some embodiments, it may be desirable to limit transfection of thepolynucleotides to certain cells or tissues. For example, the liverrepresents an important target organ for the compositions of the presentinvention in part due to its central role in metabolism and productionof proteins and accordingly diseases which are caused by defects inliver-specific gene products (e.g., the urea cycle disorders) maybenefit from specific targeting of cells (e.g., hepatocytes).Accordingly, in certain embodiments of the present invention, thestructural characteristics of the target tissue may be exploited todirect the distribution of the lipid nanoparticles to such targettissues. For example, to target hepatocytes one or more of the lipidnanoparticles that comprise the blended pharmaceutical composition maybe sized such that their dimensions are smaller than the fenestrationsof the endothelial layer lining hepatic sinusoids in the liver;accordingly the one or more of such lipid nanoparticles can readilypenetrate such endothelial fenestrations to reach the targethepatocytes. Alternatively, a lipid nanoparticle may be sized such thatthe dimensions of the liposome are of a sufficient diameter to limit orexpressly avoid distribution into certain cells or tissues. For example,one or both of the first and a second lipid nanoparticle that comprisethe blended pharmaceutical composition may be sized such that theirdimensions are larger than the fenestrations of the endothelial layerlining hepatic sinusoids to thereby limit distribution of the liposomallipid nanoparticle to hepatocytes. In such an embodiment, largeliposomal lipid nanoparticles will not easily penetrate the endothelialfenestrations, and would instead be cleared by the macrophage Kupffercells that line the liver sinusoids. Sizing of, for example, the firstand second lipid nanoparticles comprising the blended composition maytherefore provide an opportunity to further manipulate and preciselycontrol the degree to which expression of the encapsulatedpolynucleotides may be enhanced in one or more target cells. Generally,the size of at least one of the lipid nanoparticles that comprise theblended pharmaceutical composition is within the range of about 25 to250 nm, preferably less than about 250 nm, 175 nm, 150 nm, 125 nm, 100nm, 75 nm, 50 nm, 25 nm or 10 nm.

Similarly, the compositions of the present invention may be prepared topreferentially distribute to other target tissues, cells or organs, suchas the heart, lungs, kidneys, spleen. For example, the lipidnanoparticles of the present invention may be prepared to achieveenhanced delivery to the target cells and tissues. Accordingly, thecompositions of the present invention may be enriched with additionalcationic, non-cationic and PEG-modified lipids to further target tissuesor cells.

In some embodiments, the lipid nanoparticles that comprise the blendedcompositions distribute to the cells and tissues of the liver to enhancethe delivery, transfection and the subsequent expression of thepolynucleotides (e.g., mRNA) encapsulated therein by the cells andtissues of the liver (e.g., hepatocytes) to, in certain embodiments,thereby enhance the production of a functional polypeptide encoded bysuch encapsulated polynucleotide. While such compositions maypreferentially distribute into the cells and tissues of the liver, thetherapeutic effects of the expressed polynucleotides and/or thepolypeptides produced need not be limited to the target cells andtissues. For example, the targeted hepatocytes may function as a“reservoir” or “depot” capable of expressing and/or producing, andsystemically or peripherally excreting a functional protein or enzyme.Accordingly, in certain embodiments of the present invention the one ormore of the lipid nanoparticles that comprise the blended compositionmay target hepatocyes and/or preferentially distribute to the cells andtissues of the liver upon delivery. Following the transfection of thetarget hepatocytes by the polynucleotide encapsulated in one or more ofthe lipid nanoparticles that comprise the blended composition, suchpolynucleotides are expressed (e.g., translated) and a functionalproduct (e.g., a polypeptide or protein) is excreted and systemicallydistributed, where such functional product may exert a desiredtherapeutic effect.

The polynucleotides encapsulated in one or more of the lipidnanoparticles that comprise the blended composition can be delivered toand/or transfect targeted cells or tissues. In some embodiments, theencapsulated polynucleotides are capable of being expressed andfunctional polypeptide products produced (and in some instancesexcreted) by the target cell, thereby conferring a beneficial propertyto, for example the target cells or tissues. Such encapsulatedpolynucleotides may encode, for example, a hormone, enzyme, receptor,polypeptide, peptide or other protein of interest. In certainembodiments, such encapsulated polynucleotides may also encode a smallinterfering RNA (siRNA) or antisense RNA for the purpose of decreasingor eliminating expression of an endogenous nucleic acid or gene. Incertain embodiments such encapsulated polynucleotides may be natural orrecombinant in nature and may exert their therapeutic activity usingeither sense or antisense mechanisms of action.

It should be understood that while certain embodiments described hereinmay cause an enhanced or modulated expression of one or moreencapsulated polynucleotides by a target cell, the utility of theblended compositions described herein are not limited to increases inexpression of encapsulated polynucleotides. Rather, in certainembodiments, the blended compositions described herein may modulate orotherwise cause a synergistic reduction in the expression of one or moregenes or nucleic acids in a target cell. For example, in certainembodiments where one or more of the encapsulated polynucleotidescomprising the blended formulations hereof are antisenseoligonucleotides, such blended formulations may synergistically enhance(e.g., increase) the delivery of such encapsulated antisenseoligonucleotides to one or more target cells, thereby modulating orenhancing the interference with or inhibition of the expression orproduction of a targeted gene or nucleic acid. Accordingly, where theencapsulated polynucleotides are, for example, antisenseoligonucleotides or siRNA, the blended formulations comprising suchpolynucleotides may synergistically enhance the delivery of encapsulatedpolynucleotides to the target cells (e.g., enhanced by about one-, two-,three-, four-, five-, six-, eight-, ten-, twelve-, fifteen-, twenty-,twenty-five, fifty-, one-hundred, five-hundred, one thousand-fold, ormore). Such enhanced delivery of, for example, the antisense or siRNApolynucleotides, using the blended formulations described herein wouldthereby synergistically reduce the expression of the targeted nucleicacids (e.g., such that the production of a nucleic acid or proteincorresponding to such targeted nucleic acids is reduced or otherwiseeliminated). In such an embodiment, the delivery of the encapsulatedpolynucleotides to the target cells may be synergistically enhanced(e.g., increased), and accordingly the function of the encapsulatedpolynucleotides also enhanced, thereby causing a corresponding reductionin the expression of the targeted genes or nucleic acids.

Similarly, in certain embodiments, the inventions described herein maycause a synergistically enhanced (e.g., increased) production of one ormore polypeptides or proteins that are encoded by the encapsulatedpolynucleotides. In certain embodiments where such polypeptides orproteins are excreted into the peripheral circulation of a subject, suchenhanced production of polypeptides or protein encoded by, for example,an encapsulate mRNA polynucleotide, using the blended formulationsdescribed herein would thereby be expected to cause a correspondingenhanced (e.g., increased) secretion of such polypeptides peripherally.

In some embodiments, the encapsulated polynucleotides (e.g., mRNAencoding a deficient protein) may optionally include chemical orbiological modifications which, for example, improves the stabilityand/or half-life of such polynucleotide or which improves or otherwisefacilitates translation of such polynucleotide.

Also contemplated by the present invention is the co-delivery of one ormore unique polynucleotides to target cells by the lipid nanoparticlesthat comprises the blended compositions, for example, by combining twounique polynucleotides into a single lipid nanoparticle. In certainembodiments, a first polynucleotide, such as mRNA encodinggalactose-1-phosphate uridyltransferase (GALT) as represented by SEQ IDNO: 2, and a second polynucleotide, such as mRNA encoding galatokinase(GALK), may be encapsulated into a single liposomal-based lipidnanoparticle (e.g., a first lipid nanoparticle) and blended with asecond lipid nanoparticle and administered to a subject in need thereof(e.g., for the treatment of galactosemia). Alternatively, in certainembodiments, a first polynucleotide, such as mRNA encodinggalactose-1-phosphate uridyltransferase (GALT) as represented by SEQ IDNO: 2, and a second polynucleotide, such as mRNA encoding galatokinase(GALK), may be respectively encapsulated into a first and a second lipidnanoparticle, and such first and second lipid nanoparticles blended andadministered to a subject in need thereof (e.g., for the treatment ofgalactosemia). Also contemplated are the co-delivery and/orco-administration of a first polynucleotide and a second polynucleotidein a blended pharmaceutical composition. For example, such first andsecond polynucleotides (e.g., exogenous or synthetic mRNA) may berespectively encapsulated in a first and second lipid nanoparticle andsuch first and second lipid nanoparticles blended into a singlepharmaceutical composition. In certain embodiments, the secondpolynucleotide encapsulated by the second lipid nanoparticle enhancesthe delivery or enhances the expression of the first polynucleotide.Similarly, in certain embodiments, the first polynucleotide encapsulatedby the first lipid nanoparticle facilitates the delivery orsynergistically enhances the expression of the second polynucleotide.Alternatively, a polynucleotide may be encapsulated in a first lipidnanoparticle and subsequently blended with a second empty lipidnanoparticle (i.e., a lipid nanoparticle that does not encapsulate apolynucleotide). In such an embodiment, the expression of thepolynucleotide may be enhanced (e.g., increased) relative to theexpression of the polynucleotides observed when the first lipidnanoparticle is administered independently of the second lipidnanoparticle (e.g., expression of the polynucleotide may besynergistically increased by about two-, four-, five-, ten-, twelve,fifteen- or twenty-fold or more).

Also contemplated is the delivery of one or more encapsulatedpolynucleotides to one or more target cells to treat a single disorderor deficiency, wherein each such polynucleotide functions by a differentmechanism of action. For example, the blended pharmaceuticalcompositions of the present invention may comprise a firstpolynucleotide which, for example, is encapsulated in a first lipidnanoparticle and intended to correct an endogenous protein or enzymedeficiency, and which is blended with a second polynucleotideencapsulated in a second lipid nanoparticle and intended to deactivateor “knock-down” a malfunctioning endogenous polynucleotide and itsprotein or enzyme product. Such encapsulated polynucleotides may encode,for example mRNA and siRNA. Alternatively, such polynucleotides may beencapsulated in the same lipid nanoparticle and blended with an emptylipid nanoparticle. In each such embodiments, the expression of theencapsulated polynucleotides may be synergistically enhanced (e.g.,increased) relative to the expression of the polynucleotides observedwhen such first lipid nanoparticles are administered independently ofthe second lipid nanoparticles. For example, the expression of theencapsulated polynucleotide may be synergistically increased by at leastabout two-, four-, five-, ten-, twelve, fifteen- or twenty-fold or more,relative to the sum of the expression of the polynucleotides observedwhen such first lipid nanoparticles are administered independently ofthe second lipid nanoparticles.

While in vitro transcribed polynucleotides (e.g., mRNA) may betransfected into target cells, such polynucleotides may be readily andefficiently degraded by the cell in vivo, thus rendering suchpolynucleotides ineffective. Moreover, some polynucleotides are unstablein bodily fluids (particularly human serum) and can be degraded ordigested even before reaching a target cell. In addition, within a cell,a natural mRNA can decay with a half-life of between 30 minutes andseveral days. Accordingly, in certain embodiments, the lipidnanoparticle-encapsulated polynucleotides provided herein, and inparticular the mRNA polynucleotides provided herein, preferably retainat least some ability to be expressed or translated, to thereby producea functional protein or enzyme within one or more target cells.

In certain embodiments, the blended pharmaceutical compositions compriseone or more lipid nanoparticles that include or encapsulate one or morestabilized polynucleotides (e.g., mRNA which has been stabilized againstin vivo nuclease digestion or degradation) that modulate the expressionof a gene or that may be expressed or translated to produce a functionalpolypeptide or protein within one or more target cells. In certainembodiments, the activity of such encapsulated polynucleotides (e.g.,mRNA encoding a functional protein or enzyme) is prolonged over anextended period of time. For example, the activity of thepolynucleotides may be prolonged such that the blended pharmaceuticalcompositions may be administered to a subject on a semi-weekly orbi-weekly basis, or more preferably on a monthly, bi-monthly, quarterlyor an annual basis. The extended or prolonged activity of the blendedpharmaceutical compositions of the present invention, and in particularof the encapsulated mRNA, is directly related to the quantity offunctional protein or enzyme translated from such mRNA. Similarly, theactivity of the blended compositions of the present invention may befurther extended or prolonged by chemical modifications made to furtherimprove or enhance translation of the mRNA polynucleotides. For example,the Kozac consensus sequence plays a role in the initiation of proteintranslation, and the inclusion of such a Kozac consensus sequence in theencapsulated mRNA polynucleotides may further extend or prolong theactivity of the mRNA polynucleotides. Furthermore, the quantity offunctional protein or enzyme expressed and produced by the target cellis a function of the quantity of polynucleotide (e.g., mRNA) deliveredto the target cells and the stability of such polynucleotide. To theextent that the stability of the polynucleotides of the presentinvention may be improved or enhanced, the half-life, the activity ofthe translated protein or enzyme and the dosing frequency of thecomposition may be further extended.

In some embodiments, the polynucleotides encapsulated in thepharmaceutical compositions comprise mRNA (e.g., SEQ ID NO: 3 encodinghuman erythropoietin mRNA). In certain embodiments the polynucleotidescan be chemically modified for example, to confer stability (e.g.,stability relative to the wild-type or naturally-occurring version ofthe mRNA and/or the version of the mRNA naturally endogenous to targetcells). Accordingly, in some embodiments, the encapsulatedpolynucleotides provided herein comprise at least one chemicalmodification which confers increased or enhanced stability to thepolynucleotide, including, for example, improved resistance to nucleasedigestion in vivo. As used herein, the phrases “chemical modifications”and “chemically modified” as such terms relate to the polynucleotidesprovided herein, include at least one alteration which preferablyenhances stability and renders the polynucleotide more stable (e.g.,resistant to nuclease digestion) than the wild-type or naturallyoccurring version of that polynucleotide. The terms “stable” and“stability” as such terms relate to the polynucleotides of the presentinvention, and particularly with respect to the mRNA, refer to increasedor enhanced resistance to degradation by, for example nucleases (i.e.,endonucleases or exonucleases) which are normally capable of degradingsuch RNA. Increased stability can include, for example, less sensitivityto hydrolysis or other destruction by endogenous enzymes (e.g.,endonucleases or exonucleases) or conditions within the target cell ortissue, thereby increasing or enhancing the residence of suchpolynucleotides in the target cell, tissue, subject and/or cytoplasm.The stabilized polynucleotide molecules provided herein demonstratelonger half-lives relative to their naturally occurring, unmodifiedcounterparts (e.g. the wild-type version of the polynucleotide).

Also contemplated by the phrases “chemical modification” and “chemicallymodified” as such terms related to the polynucleotides of the presentinvention are alterations which improve or enhance translation of mRNApolynucleotides, including for example, the inclusion of sequences whichfunction in the initiation of protein translation (e.g., the Kozacconsensus sequence). (Kozak, M., Nucleic Acids Res 15 (20): 8125-48(1987)). The phrase “chemical modifications” as used herein, alsoinclude modifications which introduce chemistries which differ fromthose seen in naturally occurring polynucleotides, for example, covalentmodifications such as the introduction of modified nucleotides, (e.g.,nucleotide analogs, or the inclusion of pendant groups which are notnaturally found in such polynucleotide molecules). In some embodiments,the polynucleotides have undergone a chemical or biological modificationto render them more stable prior to encapsulation in one or more lipidnanoparticles. Exemplary chemical modifications to a polynucleotideinclude the depletion of a base (e.g., by deletion or by thesubstitution of one nucleotide for another) or chemical modification ofa base.

In addition, suitable modifications include alterations in one or morenucleotides of a codon such that the codon encodes the same amino acidbut is more stable than the codon found in the wild-type version of thepolynucleotide. For example, an inverse relationship between thestability of RNA and a higher number cytidines (C's) and/or uridines(U's) residues has been demonstrated, and RNA devoid of C and U residueshave been found to be stable to most RNases (Heidenreich, et al. J BiolChem 269, 2131-8 (1994)). In some embodiments, the number of C and/or Uresidues in an mRNA sequence is reduced. In a another embodiment, thenumber of C and/or U residues is reduced by substitution of one codonencoding a particular amino acid for another codon encoding the same ora related amino acid. Contemplated modifications to the mRNApolynucleotides of the present invention also include the incorporationof pseudouridines. The incorporation of pseudouridines into the mRNApolynucleotides of the present invention may enhance stability andtranslational capacity, as well as diminishing immunogenicity in vivo.(See, e.g., Karikó, K., et al., Molecular Therapy 16 (11): 1833-1840(2008)). Substitutions and modifications to the polynucleotides of thepresent invention may be performed by methods readily known to one orordinary skill in the art.

The constraints on reducing the number of C and U residues in a sequencewill likely be greater within the coding region of an mRNA, compared toan untranslated region, (i.e., it will likely not be possible toeliminate all of the C and U residues present in the message while stillretaining the ability of the message to encode the desired amino acidsequence). The degeneracy of the genetic code, however presents anopportunity to allow the number of C and/or U residues that are presentin the sequence to be reduced, while maintaining the same codingcapacity (i.e., depending on which amino acid is encoded by a codon,several different possibilities for modification of RNA sequences may bepossible). For example, the codons for Gly can be altered to GGA or GGGinstead of GGU or GGC.

The encapsulated polynucleotides may include both naturally occurring awell as non-naturally occurring variants, and accordingly suchpolynucleotides may comprise not only the known purine and pyrimidineheterocycles but also heterocyclic analogues and tautomeres thereof.Contemplated examples include, but are not limited to adenine, guanine,cytosine, thymidine, uracil, xanthine, hypoxanthine, pseudouridine,5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil,5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine,and 2-chloro-6-aminopurine. In some embodiments, at least one of thenucleotides present in the polynucleotide is a modified nucleobaseselected from the group consisting of 5-methylcytosine, isocytosine,pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine,2-aminopurine, inosine, diaminopurine, and 2-chloro-6-aminopurine.

The term chemical modification also includes, for example, theincorporation of non-nucleotide linkages or modified nucleotides intothe polynucleotide sequences of the present invention (e.g.,end-blocking modifications to one or both the 3′ and 5′ ends of an mRNAmolecule encoding a functional protein or enzyme). Such modificationsmay include the addition of bases to a polynucleotide sequence (e.g.,the inclusion of a poly A tail or a longer poly A tail), the alterationof the 3′ UTR or the 5′ UTR, complexing the polynucleotide with an agent(e.g., a protein or a complementary polynucleotide molecule), andinclusion of elements which change the structure of a polynucleotidemolecule (e.g., which form secondary structures).

The poly A tail is thought to stabilize natural messengers and syntheticsense RNA. Therefore, in certain embodiments a long poly A tail can beadded to an mRNA molecule thus rendering the RNA more stable. Poly Atails can be added using a variety of art-recognized techniques. Forexample, long poly A tails can be added to synthetic or in vitrotranscribed RNA using poly A polymerase (Yokoe, et al. NatureBiotechnology. 1996; 14: 1252-1256). A transcription vector can alsoencode long poly A tails. In addition, poly A tails can be added bytranscription directly from PCR products. Poly A may also be ligated tothe 3′ end of a sense RNA with RNA ligase (see, e.g., Molecular CloningA Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis(Cold Spring Harbor Laboratory Press: 1991 edition)). In certainembodiments, the length of the poly A tail is at least about 90, 200,300, 400 at least 500 nucleotides. In certain embodiments, the length ofthe poly A tail is adjusted to control the stability of a modified sensemRNA molecule of the invention and, thus, the transcription of protein.For example, since the length of the poly A tail can influence thehalf-life of a sense mRNA molecule, the length of the poly A tail can beadjusted to modify the level of resistance of the mRNA to nucleases andthereby control the time course of polynucleotide expression and/orpolypeptide production in a target cell. In certain embodiments, thestabilized polynucleotide molecules are sufficiently resistant to invivo degradation (e.g., by nucleases), such that they may be deliveredto the target cell without a lipid nanoparticle.

In certain embodiments, the chemical modifications are end-blockingmodification of the one or more polynucleotides which comprise theblended pharmaceutical compositions of the invention. For example, suchpolynucleotides can be modified by the incorporation 3′ and/or 5′untranslated (UTR) sequences which are not naturally found in thewild-type polynucleotide. In certain embodiments, 3′ and/or 5′ flankingsequence which naturally flanks an mRNA and encodes a second, unrelatedprotein can be incorporated into the nucleotide sequence of an mRNAmolecule encoding a or functional protein in order to modify it. Forexample, 3′ or 5′ sequences from mRNA molecules which are stable (e.g.,globin, actin, GAPDH, tubulin, histone, or citric acid cycle enzymes)can be incorporated into the 3′ and/or 5′ region of a sense mRNApolynucleotide molecule to increase the stability of the sense mRNAmolecule.

Also contemplated by the present invention are modifications to thepolynucleotide sequences made to one or both of the 3′ and 5′ ends ofthe polynucleotide. For example, the present invention contemplatesmodifications to the 5′ end of the polynucleotides (e.g., mRNA) toinclude a partial sequence of a CMV immediate-early 1 (IE1) gene, or afragment thereof to improve the nuclease resistance and/or improve thehalf-life of the polynucleotide. In addition to increasing the stabilityof the mRNA polynucleotide sequence, it has been surprisingly discoveredthe inclusion of a partial sequence of a CMV immediate-early 1 (IE1)gene (e.g., to the 5′ untranslated region of the mRNA) further enhancesthe translation of the mRNA. Also contemplated is the inclusion of asequence encoding human growth hormone (hGH), or a fragment thereof tothe 3′ end or untranslated region of the polynucleotide (e.g., mRNA) tofurther stabilize the polynucleotide. Generally, the contemplatedchemical modifications improve the stability and/or pharmacokineticproperties (e.g., half-life) of the polynucleotide relative to theirunmodified counterparts, and include, for example modifications made toimprove such polynucleotides' resistance to in vivo nuclease digestion.

Contemplated chemical modification also include, for example, modifyingencapsulated polynucleotides to include non-naturally occurringnucleotides comprising, for example modified sugar and/or base moieties,which are also referred to herein as “nucleotide analogues”.Non-naturally occurring nucleotides include nucleotides which havemodified sugar moieties, such as bicyclic nucleotides or 2′ modifiednucleotides, such as 2′ substituted nucleotides. In some embodiments,the nucleotide analogues are variants of natural nucleotides, such asDNA or RNA nucleotides, by virtue of, for example, modifications in thesugar and/or base moieties.

In certain embodiments, the contemplated nucleotide analogues may befunctionally equivalent to the naturally occurring nucleotides in thecontext of the polynucleotide. For example, the nucleotide analogues mayhave no functional effect on the way the polynucleotide functions. Suchfunctionally equivalent nucleotide analogues may nevertheless be usefulif, for example, they are easier or cheaper to manufacture, or are morestable to storage or manufacturing conditions, or represent a tag orlabel.

In other embodiments, the nucleotide analogues may have a functionaleffect on the way in which the polynucleotide functions (e.g., byincreasing resistance to intracellular nucleases and/or increased easeof transport into the target cell). Specific examples of contemplatednucleotide analogues are described by, for example, in Freier, et al.,Nucl. Acid Res. (1997) 25: 4429-4443 and Uhlmann, et al., Curr. Opinionin Drug Development (2000) 3(2): 293-213.

The polynucleotides disclosed herein may thus comprise or consist of asequence of naturally-occurring nucleotides, (e.g., DNA or mRNA), oralternatively may comprise or consist of a combination of such naturallyoccurring nucleotides and one or more non-naturally occurringnucleotides, (e.g., nucleotide analogues). In certain embodiments, forexample, where the encapsulated polynucleotides comprises or consists ofantisense oligonucleotides, the inclusion of nucleotide analogues insuch polynucleotides may suitably enhance the affinity of thepolynucleotide for one or more target sequences. Additional examples ofsuitable and preferred nucleotide analogues are provided inInternational Patent Application WO 2007/031091, the contents of whichare incorporated by reference herein.

In some embodiments the nucleotide analogues are independently selectedfrom, for example: 2′-O-alkyl-RNA units, 2′-amino-DNA units,2′-fluoro-DNA units, locked nucleic acid units, arabino nucleic acid(ANA) units, 2′-fluoro-ANA units, HNA units, INA (intercalating nucleicacid units as discussed by Christensen, et al., Nucl. Acids. Res. (2002)30: 4918-4925) and 2′MOE units. In certain embodiments there is only oneof the above types of nucleotide analogues present in thepolynucleotides of the invention.

In some embodiments the nucleotide analogues comprise2′-O-methoxyethyl-RNA (2′MOE), 2′-fluoro-DNA monomers or LNA nucleotideanalogues, and as such the polynucleotides of the invention may comprisenucleotide analogues which are independently selected from these threetypes of analogues, or alternatively may comprise only one type ofanalogue selected from the three types. In some embodiments at least oneof the nucleotides of an encapsulated polynucleotide is 2′-MOE-RNA, suchas 2, 3, 4, 5, 6, 7, 8, 9 or 10 2′-MOE-RNA nucleotide units. In someembodiments at least one of the nucleotides of an encapsulatedpolynucleotide is 2′-fluoro DNA, such as 2, 3, 4, 5, 6, 7, 8, 9 or 102′-fluoro-DNA nucleotide units.

In some embodiments, the encapsulated polynucleotides comprise at leastone locked nucleic acid (LNA) unit, such as 1, 2, 3, 4, 5, 6, 7, or 8LNA units, such as from about 3-7 or 4-8 LNA units, or 3, 4, 5, 6 or 7LNA units. In some embodiments, all the nucleotides of thepolynucleotide_are LNA. In some embodiments, the polynucleotide maycomprise both beta-D-oxy-LNA, and one or more of the following LNAunits: thio-LNA, amino-LNA, oxy-LNA, and/or ENA in either the beta-D oralpha-L configurations or combinations thereof. In some embodiments allLNA cytosine units are 5′methyl-cytosine.

In some embodiments, the blended pharmaceutical composition, the two ormore lipid nanoparticles comprised therein or the polynucleotidesencapsulated by such lipid nanoparticles can comprise a stabilizingreagent. The compositions can include one or more formulation reagentsthat bind directly or indirectly to, and stabilize the polynucleotide,thereby enhancing residence time in the cytoplasm of a target cell. Suchreagents preferably lead to an improved half-life of a polynucleotide inthe target cells. For example, the stability of an mRNA and efficiencyof translation may be increased by the incorporation of “stabilizingreagents” that form complexes with the polynucleotides (e.g., mRNA) thatnaturally occur within a cell (see e.g., U.S. Pat. No. 5,677,124).Incorporation of a stabilizing reagent can be accomplished for example,by combining the poly A and a protein with the mRNA to be stabilized invitro before loading or encapsulating the mRNA within the one or morelipid nanoparticles that comprise the blended pharmaceuticalcomposition. Exemplary stabilizing reagents include one or moreproteins, peptides, aptamers, translational accessory protein, mRNAbinding proteins, and/or translation initiation factors.

Stabilization of the blended pharmaceutical compositions describedherein, and of the constituent lipid nanoparticles, may also be improvedby the use of opsonization-inhibiting moieties, which are typicallylarge hydrophilic polymers that are chemically or physically bound orotherwise incorporated into the lipid nanoparticle (e.g., by theintercalation of a lipid-soluble anchor into the membrane itself, or bybinding directly to active groups of membrane lipids). Theseopsonization-inhibiting hydrophilic polymers form a protective surfacelayer which significantly decreases the uptake of the liposomes by themacrophage-monocyte system and reticulo-endothelial system (e.g., asdescribed in U.S. Pat. No. 4,920,016, the entire disclosure of which isherein incorporated by reference). For example, delays in the uptake oflipid nanoparticles by the reticuloendothelial system may be facilitatedby the addition of a hydrophilic polymer surface coating onto or intolipid nanoparticles to mask the recognition and uptake of theliposomal-based lipid nanoparticle by the reticuloendothelial system.For example, in certain embodiments, one or more of the lipidnanoparticles that comprise the blended formulations comprise apolyethyleneglycol (PEG) polymer or a PEG-modified lipid to furtherenhance delivery of such lipid nanoparticles to the target cell andtissues.

When RNA is hybridized to a complementary polynucleotide molecule (e.g.,DNA or RNA) it may be protected from nucleases. (Krieg, et al. Melton.Methods in Enzymology. 1987; 155, 397-415). The stability of hybridizedmRNA is likely due to the inherent single strand specificity of mostRNases. In some embodiments, the stabilizing reagent selected to complexa polynucleotide is a eukaryotic protein, (e.g., a mammalian protein).In yet another embodiment, the polynucleotide (e.g., mRNA) for use insense therapy can be modified by hybridization to a secondpolynucleotide molecule. If an entire mRNA molecule were hybridized to acomplementary polynucleotide molecule translation initiation may bereduced. In some embodiments the 5′ untranslated region and the AUGstart region of the mRNA molecule may optionally be left unhybridized.Following translation initiation, the unwinding activity of the ribosomecomplex can function even on high affinity duplexes so that translationcan proceed. (Liebhaber. J. Mol. Biol. 1992; 226: 2-13; Monia, et al. JBiol Chem. 1993; 268: 14514-22.) It will be understood that any of theabove described methods for enhancing the stability of polynucleotidesmay be used either alone or in combination with one or more of any ofthe other above-described methods and/or compositions.

In certain embodiments, the blended pharmaceutical compositions of thepresent invention enhance the delivery of lipidnanoparticle-encapsulated polynucleotides to one or more target cells,tissues or organs. In some embodiments, enhanced delivery to one or moretarget cells comprises increasing the amount of polynucleotide thatcomes in contact or is otherwise delivered to the target cells. In someembodiments, enhancing delivery to target cells comprises reducing theamount of polynucleotide that comes into contact with non-target cells.In some embodiments, enhancing delivery to target cells comprisesallowing the transfection of at least some target cells with theencapsulated polynucleotide. In some embodiments, the level ofexpression of the polynucleotide encapsulated by the lipid nanoparticleswhich comprise the subject blended pharmaceutical compositions isincreased in target cells.

The polynucleotides of the present invention may be optionally combinedwith a reporter gene (e.g., upstream or downstream of the coding regionof the polynucleotide) which, for example, facilitates the determinationof polynucleotide delivery to the target cells or tissues. Suitablereporter genes may include, for example, Green Fluorescent Protein mRNA(GFP mRNA), Renilla Luciferase mRNA (Luciferase mRNA), FireflyLuciferase mRNA (SEQ ID NO: 1), or any combinations thereof. Forexample, GFP mRNA may be fused with a polynucleotide encoding OTC mRNAto facilitate confirmation of mRNA localization in the target cells,tissues or organs.

In some embodiments, the blended compositions of the present inventioncomprise one or more additional molecules (e.g., proteins, peptides,aptamers or oliogonucleotides) which facilitate the transfer of thepolynucleotides (e.g., mRNA, miRNA, snRNA and snoRNA) from the lipidnanoparticle into an intracellular compartment of the target cell. Insome embodiments, the additional molecule facilitates the delivery ofthe polynucleotides into, for example, the cytosol, the lysosome, themitochondrion, the nucleus, the nucleolae or the proteasome of a targetcell. Also included are agents that facilitate the transport of thetranslated protein of interest from the cytoplasm to its normalintercellular location (e.g., in the mitochondrion) to treatdeficiencies in that organelle. In some embodiments, the agent isselected from the group consisting of a protein, a peptide, an aptamer,and an oligonucleotide.

In some embodiments, the compositions of the present inventionfacilitate a subject's endogenous production of one or more functionalproteins and/or enzymes, and in particular the production of proteinsand/or enzymes which demonstrate less immunogenicity relative to theirrecombinantly-prepared counterparts. In a certain embodiments of thepresent invention, the lipid nanoparticles comprise polynucleotideswhich encode mRNA of a deficient protein or enzyme. Upon distribution ofsuch compositions to the target tissues and the subsequent transfectionof such target cells, the exogenous mRNA loaded or encapsulated into thelipid nanoparticles that comprise the blended compositions may betranslated in vivo to produce a functional protein or enzyme encoded bysuch encapsulated mRNA (e.g., a protein or enzyme in which the subjectis deficient). Accordingly, in certain embodiments the compositions ofthe present invention exploit a subject's ability to translateexogenously- or recombinantly-prepared mRNA to produce anendogenously-translated protein or enzyme, and thereby produce (andwhere applicable excrete) a functional protein or enzyme. The expressedmRNA and/or translated proteins or enzymes produced therefrom may alsobe characterized by the in vivo inclusion of native post-translationalmodifications which may often be absent in recombinantly-preparedproteins or enzymes, thereby further reducing the immunogenicity of thetranslated protein or enzyme.

The encapsulation of mRNA in the lipid nanoparticles and theadministration of the blended pharmaceutical compositions comprisingsuch lipid nanoparticles avoids the need to deliver the mRNA to specificorganelles within a target cell (e.g., mitochondria). Rather, upontransfection of a target cell and delivery of the encapsulated mRNA tothe cytoplasm of the target cell, the mRNA contents of the lipidnanoparticles may be translated and a functional protein or enzymeproduced and/or excreted.

The present invention also contemplates the discriminatory targeting ofone or more target cells and tissues by both passive and activetargeting means. The phenomenon of passive targeting exploits thenatural distributions patterns of lipid nanoparticles in vivo withoutrelying upon the use of additional excipients or means to enhancerecognition of the lipid nanoparticle by one or more target cells. Forexample, lipid nanoparticles which are subject to phagocytosis by thecells of the reticulo-endothelial system are likely to accumulate in theliver or spleen, and accordingly may provide means to passively directthe delivery of the compositions to such target cells.

Alternatively, the present invention contemplates active targeting,which involves the use of additional excipients, referred to herein as“targeting ligands” that may be bound (either covalently ornon-covalently) to the lipid nanoparticle to encourage localization ofsuch lipid nanoparticle at certain target cells or target tissues. Forexample, targeting may be mediated by the inclusion of one or moreendogenous targeting ligands (e.g., apolipoprotein E) in or on the lipidnanoparticle to encourage distribution to the target cells or tissues.Recognition of the targeting ligand by the target tissues activelyfacilitates tissue distribution to, and cellular uptake of the lipidnanoparticles and/or their contents by the target cells and tissues. Forexample, in certain embodiments, one or more of the lipid nanoparticlesthat comprise the blended pharmaceutical formulation may comprise anapolipoprotein-E targeting ligand in or on such lipid nanoparticles tofacilitate or encourage recognition and binding of such lipidnanoparticle to endogenous low density lipoprotein receptors expressed,for example by hepatocytes. As provided herein, the composition cancomprise a ligand capable of enhancing affinity of the blendedcompositions to one or more target cells. Targeting ligands may belinked to the outer bilayer of the lipid nanoparticle during formulationor post-formulation. These methods are well known in the art. Inaddition, some lipid nanoparticles may comprise fusogenic polymers suchas PEAA, hemagluttinin, other lipopeptides (see U.S. patent applicationSer. No. 08/835,281, and 60/083,294, which are incorporated herein byreference) and other features useful for in vivo and/or intracellulardelivery. In other embodiments, the blended compositions of the presentinvention demonstrate improved transfection efficacies, and/ordemonstrate enhanced selectivity towards target cells or tissues ofinterest. Contemplated therefore are blended compositions or lipidnanoparticles that comprise one or more ligands (e.g., peptides,aptamers, oligonucleotides, a vitamin or other molecules) that arecapable of enhancing the affinity of the blended compositions or theirconstituent lipid nanoparticles and their polynucleotide contents to oneor more target cells or tissues. Suitable ligands may optionally bebound or linked to the surface of the lipid nanoparticle. In someembodiments, the targeting ligand may span the surface of a lipidnanoparticle or be encapsulated within the lipid nanoparticle. Suitableligands are selected based upon their physical, chemical or biologicalproperties (e.g., selective affinity and/or recognition of target cellsurface markers or features.) Cell-specific target sites and theircorresponding targeting ligand can vary widely. Suitable targetingligands are selected such that the unique characteristics of a targetcell are exploited, thus allowing the composition to discriminatebetween target and non-target cells. For example, compositions of thepresent invention may bear surface markers (e.g., apolipoprotein-B orapolipoprotein-E) that selectively enhance recognition of, or affinityto hepatocytes (e.g., by receptor-mediated recognition of and binding tosuch surface markers). Additionally, the use of galactose as a targetingligand would be expected to direct the compositions of the presentinvention to parenchymal hepatocytes, or alternatively the use ofmannose containing sugar residues as a targeting ligand would beexpected to direct the compositions of the present invention to liverendothelial cells (e.g., mannose containing sugar residues that may bindpreferentially to the asialoglycoprotein receptor present inhepatocytes). (See Hillery A M, et al. “Drug Delivery and Targeting: ForPharmacists and Pharmaceutical Scientists” (2002) Taylor & Francis,Inc.) The presentation of such targeting ligands that have beenconjugated to moieties present in the lipid nanoparticle thereforefacilitate recognition and uptake of the blended compositions of thepresent invention by one or more target cells and tissues. Examples ofsuitable targeting ligands include one or more peptides, proteins,aptamers, vitamins and oligonucleotides.

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to, humans, non-human primates,rodents, and the like, to which the blended compositions and methods ofthe present invention may be administered. Typically, the terms“subject” and “patient” are used interchangeably herein in reference toa human subject.

The ability of the blended lipid nanoparticle compositions tosynergistically enhance the expression of encapsulated polynucleotidesas the production of a polypeptide or protein provides novel and moreefficient means of effectuating the in vivo production of polypeptidesand proteins for the treatment of a host of diseases or pathologicalconditions. Such blended lipid nanoparticle compositions areparticularly suitable for the treatment of diseases or pathologicalconditions associated with the aberrant expression of a protein orenzyme. For example, the successful delivery of polynucleotides such asmRNA to target organs such as the liver and in particular, tohepatocytes, can be used for the treatment and the correction of in-bornerrors of metabolism that are localized to the liver. Accordingly, theblended pharmaceutical compositions and related methods described hereinmay be employed to treat a wide range of diseases and pathologicalconditions, in particular those diseases which are due to protein orenzyme deficiencies. The polynucleotides encapsulated by the lipidnanoparticles that comprise the blended pharmaceutical compositions mayencode a functional product (e.g., a protein, enzyme, polypeptide,peptide and/or functional RNA), and may encodes a product whose in vivoproduction is desired. Alternatively, the polynucleotides encapsulatedby the lipid nanoparticles that comprise the blended pharmaceuticalcompositions may comprise an antisense oligonucleotide and followingdelivery of such antisense oligonucleotide to one or more target cells,the expression of targeted genes or nucleic acids modulated,synergistically reduced or eliminated.

The urea cycle metabolic disorders represent examples of such proteinand enzyme deficiencies which may be treated using the methods andblended lipid nanoparticle compositions provided herein. Such urea cyclemetabolic disorders include ornithine transcarbamylase (OTC) deficiency,arginosuccinate synthetase deficiency (ASD) and argininosuccinate lyasedeficiency (ALD). Therefore, in some embodiments, the polynucleotidesencapsulated by the lipid nanoparticles provided herein encode an enzymeinvolved in the urea cycle, including, for example, ornithinetranscarbamylase (OTC), carbamyl phosphate synthetase (CPS),argininosuccinate synthetase 1 (ASS1) argininosuccinate lyase (ASL), andarginase (ARG).

Five metabolic disorders which result from defects in the biosynthesisof the enzymes involved in the urea cycle have been described, andinclude ornithine transcarbamylase (OTC) deficiency, carbamyl phosphatesynthetase (CPS) deficiency, argininosuccinate synthetase 1 (ASS1)deficiency (citrullinemia), argininosuccinate lyase (ASL) deficiency andarginase deficiency (ARG). Of these five metabolic disorders, OTCdeficiency represents the most common, occurring in an estimated one outof every 80,000 births.

OTC is a homotrimeric mitochondrial enzyme which is expressed almostexclusively in the liver and which encodes a precursor OTC protein thatis cleaved in two steps upon incorporation into the mitchondrial matrix.(Horwich A L., et al. Cell 1986; 44: 451-459). OTC deficiency is agenetic disorder which results in a mutated and biologically inactiveform of the enzyme ornithine transcarbamylase. OTC deficiency oftenbecomes evident in the first few days of life, typically after proteiningestion. In the classic severe form of OTC deficiency, within thefirst days of life patients present with lethargy, convulsions, coma andsevere hyperammonemia, which quickly leads to a deteriorating and fataloutcome absent appropriate medical intervention. (Monish S., et al.,Genetics for Pediatricians; Remedica, Cold Spring Harbor Laboratory(2005)). If improperly treated or if left untreated, complications fromOTC deficiency may include developmental delay and mental retardation.OTC deficient subjects may also present with progressive liver damage,skin lesions, and brittle hair. In some affected individuals, signs andsymptoms of OTC deficiency may be less severe, and may not appear untillater in life.

The OTC gene, which is located on the short arm of the X chromosomewithin band Xp21.1, spans more than 85 kb and is comprised of 10 exonsencoding a protein of 1062 amino acids. (Lindgren V., et al. Science1984; 226: 698-7700; Horwich, A L., et al. Science 224: 1068-1074, 1984;Horwich, A L. et al., Cell 44: 451-459, 1986; Hata, A., et al., J.Biochem. 100: 717-725, 1986, which are incorporated herein byreference). The OTC enzyme catalyzes the conversion or ornithine andcarbamoyl phosphate to citrulline. Since OTC is on the X chromosome,females are primarily carriers while males with nonconservativemutations rarely survive past 72 hours of birth.

In healthy subjects, OTC is expressed almost exclusively inhepatocellular mitochondria. Although not expressed in the brain ofhealthy subjects, OTC deficiency can lead to neurological disorders. Forexample, one of the usual symptoms of OTC deficiency, which isheterogeneous in its presentation, is hyperammonaemic coma (Gordon, N.,Eur J Paediatr Neurol 2003; 7:115-121.).

OTC deficiency is very heterogeneous, with over 200 unique mutationsreported and large deletions that account for approximately 10-15% ofall mutations, while the remainder generally comprises missense pointmutations with smaller numbers of nonsense, splice-site and smalldeletion mutations. (Monish A., et al.) The phenotype of OTC deficiencyis extremely heterogeneous, which can range from acute neonatalhyperammonemic coma to asymptomatic hemizygous adults. (Gordon N. Eur JPaediatr Neurol 2003; 7: 115-121). Those mutations that result in severeand life threatening neonatal disease are clustered in importantstructural and functional domains in the interior of the protein atsites of enzyme activity or at the interchain surface, while mutationsassociated with late-onset disease are located on the protein surface(Monish A., et al.) Patients with milder or partial forms of OTCdeficiency may have onset of disease later in life, which may present asrecurrent vomiting, neurobehavioral changes or seizures associated withhyperammonemia.

The blended lipid nanoparticle compositions and related methods of thepresent invention are broadly applicable to the delivery ofpolynucleotides, and in particular mRNA, to treat a number of disorders.In particular, the blended lipid nanoparticle compositions and relatedmethods of the present invention are suitable for the treatment ofdiseases or disorders relating to the deficiency of proteins and/orenzymes. In certain embodiments, the lipid nanoparticle-encapsulatedpolynucleotides encode functional proteins or enzymes that are excretedor secreted by one or more target cells into the surroundingextracellular fluid (e.g., mRNA encoding hormones andneurotransmitters). Alternatively, in another embodiment, thepolynucleotides of the present invention encode functional proteins orenzymes that remain in the cytosol of one or more target cells (e.g.,mRNA encoding enzymes associated with urea cycle metabolic disorders oran enzyme associated with a lysosomal storage disorder). Other disordersfor which the blended lipid nanoparticle pharmaceutical compositions andrelated methods of the present invention are useful include, but are notlimited to, disorders such as SMN1-related spinal muscular atrophy(SMA); amyotrophic lateral sclerosis (ALS); GALT-related galactosemia;Cystic Fibrosis (CF); SLC3A1-related disorders including cystinuria;COL4A5-related disorders including Alport syndrome; galactocerebrosidasedeficiencies; X-linked adrenoleukodystrophy and adrenomyeloneuropathy;Friedreich's ataxia; Pelizaeus-Merzbacher disease; TSC1 and TSC2-relatedtuberous sclerosis; Sanfilippo B syndrome (MPS IIIB); CTNS-relatedcystinosis; the FMR1-related disorders which include Fragile X syndrome,Fragile X-Associated Tremor/Ataxia Syndrome and Fragile X PrematureOvarian Failure Syndrome; Prader-Willi syndrome; Fabry disease;hereditary hemorrhagic telangiectasia (AT); Niemann-Pick disease TypeCl; the neuronal ceroid lipofuscinoses-related diseases includingJuvenile Neuronal Ceroid Lipofuscinosis (JNCL), Juvenile Batten disease,Santavuori-Haltia disease, Jansky-Bielschowsky disease, and PTT-1 andTPP1 deficiencies; EIF2B1, EIF2B2, EIF2B3, EIF2B4 and EIF2B5-relatedchildhood ataxia with central nervous system hypomyelination/vanishingwhite matter; CACNA1A and CACNB4-related Episodic Ataxia Type 2; theMECP2-related disorders including Classic Rett Syndrome, MECP2-relatedSevere Neonatal Encephalopathy and PPM-X Syndrome; CDKLS-relatedAtypical Rett Syndrome; Kennedy's disease (SBMA); Notch-3 relatedcerebral autosomal dominant arteriopathy with subcortical infarcts andleukoencephalopathy (CADASIL); SCN1A and SCN1B-related seizuredisorders; the Polymerase G-related disorders which includeAlpers-Huttenlocher syndrome, POLG-related sensory ataxic neuropathy,dysarthria, and ophthalmoparesis, and autosomal dominant and recessiveprogressive external ophthalmoplegia with mitochondrial DNA deletions;X-Linked adrenal hypoplasia; X-linked agammaglobulinemia; and Wilson'sdisease. In certain embodiments, the polynucleotides, and in particularmRNA, of the present invention may encode functional proteins orenzymes. For example, the compositions of the present invention mayinclude mRNA encoding agalsidase alfa, erythropoietin, al-antitrypsin,carboxypeptidase N, alpha-L-iduronidase, iduronate-2-sulfatase,N-acetylglucosamine-1-phosphate transferase, N-acetylglucosaminidase,alpha-glucosaminide acetyltransferase, N-acetylglucosamine 6-sulfatase,N-acetylgalactosamine-4-sulfatase, beta-glucosidase, galactose-6-sulfatesulfatase, beta-galactosidase, beta-glucuronidase, glucocerebrosidase,heparan sulfamidase, hyaluronidase and galactocerebrosidase or humangrowth hormone.

Alternatively the encapsulated polynucleotides may encode full lengthantibodies or smaller antibodies (e.g., both heavy and light chains) toconfer immunity to a subject. Certain embodiments of the presentinvention relate to blended lipid nanoparticle pharmaceuticalcompositions and methods of using the same to conferring immunity to asubject (e.g., via the translation of mRNA nucleic acids encodingfunctional antibodies), the inventions disclosed herein and contemplatedhereby are broadly applicable. In an alternative embodiment the blendedcompositions of the present invention encode antibodies that may be usedto transiently or chronically effect a functional response in subjects.For example, the encapsulated mRNA may encode a functional monoclonal orpolyclonal antibody, which upon translation (and as applicable, systemicexcretion from the target cells) may be useful for targeting and/orinactivating a biological target (e.g., a stimulatory cytokine such astumor necrosis factor). Similarly, the encapsulated mRNA may encode, forexample, functional anti-nephritic factor antibodies useful for thetreatment of membranoproliferative glomerulonephritis type II or acutehemolytic uremic syndrome, or alternatively may encode anti-vascularendothelial growth factor (VEGF) antibodies useful for the treatment ofVEGF-mediated diseases, such as cancer.

The blended pharmaceutical compositions may be administered to asubject. In some embodiments, the blended compositions or theconstituent lipid nanoparticles are formulated in combination with oneor more additional polynucleotides, carriers, targeting ligands orstabilizing reagents, or in blended pharmacological compositions wheresuch compositions comprise other suitable excipients. For example, incertain embodiments, the lipid nanoparticles that comprise the blendedcompositions may be prepared to deliver nucleic acids (e.g., mRNA)encoding two or more distinct proteins or enzymes. Alternatively, theblended lipid nanoparticle compositions of the present invention may beprepared to deliver a single polypeptide in two or more lipidnanoparticles, each having distinct lipid compositions and that aresubsequently blended into a single formulation or dosage form andadministered to a subject. Techniques for formulation and administrationof drugs may be found in “Remington's Pharmaceutical Sciences,” MackPublishing Co., Easton, Pa., latest edition.

A wide range of molecules that can exert pharmaceutical or therapeuticeffects can be delivered to target cells using the blended lipidnanoparticle compositions and methods of the present invention. Themolecules can be organic or inorganic. Organic molecules can bepeptides, proteins, carbohydrates, lipids, sterols, nucleic acids(including peptide nucleic acids), or any combination thereof. Aformulation for delivery into target cells can comprise more than onetype of molecule, for example, two different polynucleotide sequencesencoding a protein, an enzyme and/or a steroid.

The blended lipid nanoparticle compositions of the present invention maybe administered and dosed in accordance with current medical practice,taking into account the clinical condition of the subject, the site andmethod of administration, the scheduling of administration, thesubject's age, sex, body weight and other factors relevant to cliniciansof ordinary skill in the art. The “effective amount” for the purposesherein may be determined by such relevant considerations as are known tothose of ordinary skill in experimental clinical research,pharmacological, clinical and medical arts, recognizing that blendedlipid nanoparticle compositions are capable of synergistically enhancingthe expression of the encapsulated polynucleotides and the production ofpolypeptides or proteins encoded thereby or modulating the expression ofa target nucleic acid or polynucleotide (e.g., using an antisenseoligonucleotide), and that in some instances dosage reductions of suchencapsulated polynucleotides relative to traditional non-blendedformulation may be warranted. In some embodiments, the amountadministered is effective to achieve at least some stabilization,improvement or elimination of symptoms and other indicators as areselected as appropriate measures of disease progress, regression orimprovement by those of skill in the art. For example, a suitable amountand dosing regimen is one that causes at least transient expression ofthe one or more polynucleotides in the target cells.

The synergistic enhancements in expression of encapsulatedpolynucleotides that characterize the blended lipid nanoparticleformulations of the present invention allow therapeutically effectiveconcentrations of polypeptides produced upon the expression of suchencapsulated polynucleotides (e.g., a therapeutic protein or enzyme) tobe achieved in the targeted tissues (or serum if the product is excretedby target cell) using a significantly lower dose of polynucleotide thanwas previously anticipated. Accordingly, in certain embodiments, theeffective amount of a polynucleotide required to achieve a desiredtherapeutic effect may be reduced by encapsulating such polynucleotidein one or more lipid nanoparticles and blending at least two lipidnanoparticles. Also contemplated are methods of reducing the amount of apolynucleotide required to elicit a therapeutic effect in a subject.Such methods generally comprise a step of administering a pharmaceuticalcomposition to the subject, wherein the pharmaceutical compositioncomprises a first lipid nanoparticle blended with a second lipidnanoparticle, and wherein one or both of the first lipid nanoparticleand the second lipid nanoparticle comprise the polynucleotide, followedby the transfection of one or more target cells of the subject with suchpolynucleotides, such that the amount of the polynucleotide required toeffectuate a therapeutic effect is reduced (e.g., reduced relative tothe amount of polynucleotide required to effectuate a therapeutic effectusing a non-blended composition or other standard techniques). Incertain embodiments, the amount of a polynucleotide required toeffectuate a therapeutic effect is reduced by at least about 10%, 15%,20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or 99%. In certainembodiments, the amount of a polynucleotide required to effectuate atherapeutic effect is reduced by at least two-, three-, four-, five-,six-, seven-, ten-, twelve-, fifteen-, twenty- or twenty-five-fold ormore.

Suitable routes of administration of the blended lipid nanoparticlecompositions include, for example, oral, rectal, vaginal, transmucosal,sublingual, subdural, nasally, or intestinal administration; parenteraldelivery, including intramuscular, subcutaneous, intramedullaryinjections, as well as intrathecal, direct intraventricular,intravenous, intraperitoneal, intranasal, opthalmically or intraocularinjections or infusions. In certain embodiments, the administration ofthe blended lipid nanoparticle composition to a subject facilitates thecontacting of the constituent lipid nanoparticles to one or more targetcells, tissues or organs.

Alternately, the blended lipid nanoparticle compositions of the presentinvention may be administered in a local rather than systemic manner,for example, via injection or infusion of the blended pharmaceuticalcomposition directly into a targeted tissue, preferably in a depot orsustained release formulation, such that the contacting of the targetedcells with the constituent lipid nanoparticles may further facilitated.Local delivery can be affected in various ways, depending on the tissueto be targeted. For example, aerosols containing compositions of thepresent invention can be inhaled (for nasal, tracheal, or bronchialdelivery); blended compositions of the present invention can be injectedinto the site of injury, disease manifestation, or pain, for example;blended compositions can be provided in lozenges for oral, tracheal, oresophageal application; can be supplied in liquid, tablet or capsuleform for administration to the stomach or intestines, can be supplied insuppository form for rectal or vaginal application; or can even bedelivered to the eye by use of creams, drops, or even injection.Formulations containing blended compositions of the present inventioncomplexed with therapeutic molecules or ligands can even be surgicallyadministered, for example in association with a polymer or otherstructure or substance that can allow the compositions to diffuse fromthe site of implantation to surrounding cells. Alternatively, suchblended compositions can be applied surgically without the use ofpolymers or supports.

In certain embodiments, the blended compositions of the presentinvention are formulated such that they are suitable forextended-release of the polynucleotides or nucleic acids encapsulated inthe constituent lipid nanoparticles. Such extended-release blendedcompositions may be conveniently administered to a subject at extendeddosing intervals. For example, in certain embodiments, the compositionsof the present invention are administered to a subject twice day, dailyor every other day. In a certain embodiments, the compositions of thepresent invention are administered to a subject twice a week, once aweek, every ten days, every two weeks, every three weeks, or morepreferably every four weeks, once a month, every six weeks, every eightweeks, every other month, every three months, every four months, everysix months, every eight months, every nine months or annually. Alsocontemplated are compositions and lipid nanoparticles which areformulated for depot administration (e.g., intramuscularly,subcutaneously, intravitreally) to either deliver or release apolynucleotide (e.g., mRNA) over extended periods of time. Preferably,the extended-release means employed are combined with modifications(e.g., chemical modifications) introduced into the polynucleotides toenhance stability.

Also contemplated herein are lyophilized pharmaceutical compositionscomprising one or more of the compounds disclosed herein and relatedmethods for the use of such lyophilized compositions as disclosed forexample, in United States Provisional Application No. PCT/US2012/041663,filed Jun. 8, 2011, the teachings of which are incorporated herein byreference in their entirety.

While certain compounds, compositions and methods of the presentinvention have been described with specificity in accordance withcertain embodiments, the following examples serve only to illustrate thecompounds of the invention and are not intended to limit the same. Eachof the publications, reference materials and the like referenced hereinto describe the background of the invention and to provide additionaldetail regarding its practice are hereby incorporated by reference intheir entirety.

The articles “a” and “an” as used herein in the specification and in theclaims, unless clearly indicated to the contrary, should be understoodto include the plural referents. Claims or descriptions that include“or” between one or more members of a group are considered satisfied ifone, more than one, or all of the group members are present in, employedin, or otherwise relevant to a given product or process unless indicatedto the contrary or otherwise evident from the context. The inventionincludes embodiments in which exactly one member of the group is presentin, employed in, or otherwise relevant to a given product or process.The invention also includes embodiments in which more than one, or theentire group members are present in, employed in, or otherwise relevantto a given product or process. Furthermore, it is to be understood thatthe invention encompasses all variations, combinations, and permutationsin which one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the listed claims is introduced into anotherclaim dependent on the same base claim (or, as relevant, any otherclaim) unless otherwise indicated or unless it would be evident to oneof ordinary skill in the art that a contradiction or inconsistency wouldarise. Where elements are presented as lists, (e.g., in Markush group orsimilar format) it is to be understood that each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should be understood that, in general, where the invention, oraspects of the invention, is/are referred to as comprising particularelements, features, etc., certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements, features, etc. For purposes of simplicity those embodimentshave not in every case been specifically set forth in so many wordsherein. It should also be understood that any embodiment or aspect ofthe invention can be explicitly excluded from the claims, regardless ofwhether the specific exclusion is recited in the specification. Thepublications and other reference materials referenced herein to describethe background of the invention and to provide additional detailregarding its practice are hereby incorporated by reference.

EXAMPLES

The following examples generally relate to lipid nanoparticlepharmaceutical compositions and formulations, and in particularpharmaceutical compositions and formulations which comprise “blends” ofsuch lipid nanoparticles, as well as highly efficacious methods of usingthe foregoing pharmaceutical compositions and formulations to deliverpolynucleotide constructs to one or more target cells, tissues andorgans.

Example 1. Formulations and Messenger RNA Material Lipid Materials

The formulations described herein include a multi-component lipidmixture of varying ratios employing one or more cationic lipids, helperlipids and PEGylated lipids designed to encapsulate various nucleicacid-based materials. Cationic lipids can include, but are not limitedto, DOTAP (1,2-dioleyl-3-trimethylammonium propane), DODAP(1,2-dioleyl-3-dimethylammonium propane), DOTMA(1,2-di-O-octadecenyl-3-trimethylammonium propane), DLinDMA (Heyes, J.;Palmer, L.; Bremner, K.; MacLachlan, I. “Cationic lipid saturationinfluences intracellular delivery of encapsulated nucleic acids” J.Contr. Rel. 2005, 107, 276-287), DLin-KC2-DMA (Semple, S. C. et al.“Rational Design of Cationic Lipids for siRNA Delivery” Nature Biotech.2010, 28, 172-176), C12-200 (Love, K. T. et al. “Lipid-like materialsfor low-dose in vivo gene silencing” PNAS 2010, 107, 1864-1869),HGT4003, ICE, dialkylamino-based, imidazole-based or guanidinium-based.Other nanoparticle components may include, but are not limited to, DSPC(1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC(1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE(1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DPPE(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE(1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG(2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)), or cholesterol. ThePEGylated lipids may include, but are not limited to, a poly(ethylene)glycol chain of up to 5 kDa in length covalently attached to a lipidwith alkyl chain(s) of C₆-C₂₀ length.

Messenger RNA Material

Codon-optimized firefly luciferase messenger RNA (CO-FFL mRNA),galactose-1-phosphate uridyl transferase (GALT) and human erythropoietin(EPO) were synthesized by in vitro transcription from a plasmid DNAtemplate encoding the gene, which was followed by the addition of a 5′cap structure (Cap1) (Fechter, P.; Brownlee, G. G. “Recognition of mRNAcap structures by viral and cellular proteins” J. Gen. Virology 2005,86, 1239-1249) and a 3′ poly(A) tail of approximately 200 nucleotides inlength as determined by gel electrophoresis. 5′ and 3′ untranslatedregions present in each mRNA product are represented as X and Y,respectively and defined as stated.

CO-FF Luciferase mRNA: (SEQ ID NO: 1)XAUGGAAGAUGCCAAAAACAUUAAGAAGGGCCCAGCGCCAUUCUACCCACUCGAAGACGGGACCGCCGGCGAGCAGCUGCACAAAGCCAUGAAGCGCUACGCCCUGGUGCCCGGCACCAUCGCCUUUACCGACGCACAUAUCGAGGUGGACAUUACCUACGCCGAGUACUUCGAGAUGAGCGUUCGGCUGGCAGAAGCUAUGAAGCGCUAUGGGCUGAAUACAAACCAUCGGAUCGUGGUGUGCAGCGAGAAUAGCUUGCAGUUCUUCAUGCCCGUGUUGGGUGCCCUGUUCAUCGGUGUGGCUGUGGCCCCAGCUAACGACAUCUACAACGAGCGCGAGCUGCUGAACAGCAUGGGCAUCAGCCAGCCCACCGUCGUAUUCGUGAGCAAGAAAGGGCUGCAAAAGAUCCUCAACGUGCAAAAGAAGCUACCGAUCAUACAAAAGAUCAUCAUCAUGGAUAGCAAGACCGACUACCAGGGCUUCCAAAGCAUGUACACCUUCGUGACUUCCCAUUUGCCACCCGGCUUCAACGAGUACGACUUCGUGCCCGAGAGCUUCGACCGGGACAAAACCAUCGCCCUGAUCAUGAACAGUAGUGGCAGUACCGGAUUGCCCAAGGGCGUAGCCCUACCGCACCGCACCGCUUGUGUCCGAUUCAGUCAUGCCCGCGACCCCAUCUUCGGCAACCAGAUCAUCCCCGACACCGCUAUCCUCAGCGUGGUGCCAUUUCACCACGGCUUCGGCAUGUUCACCACGCUGGGCUACUUGAUCUGCGGCUUUCGGGUCGUGCUCAUGUACCGCUUCGAGGAGGAGCUAUUCUUGCGCAGCUUGCAAGACUAUAAGAUUCAAUCUGCCCUGCUGGUGCCCACACUAUUUAGCUUCUUCGCUAAGAGCACUCUCAUCGACAAGUACGACCUAAGCAACUUGCACGAGAUCGCCAGCGGCGGGGCGCCGCUCAGCAAGGAGGUAGGUGAGGCCGUGGCCAAACGCUUCCACCUACCAGGCAUCCGCCAGGGCUACGGCCUGACAGAAACAACCAGCGCCAUUCUGAUCACCCCCGAAGGGGACGACAAGCCUGGCGCAGUAGGCAAGGUGGUGCCCUUCUUCGAGGCUAAGGUGGUGGACUUGGACACCGGUAAGACACUGGGUGUGAACCAGCGCGGCGAGCUGUGCGUCCGUGGCCCCAUGAUCAUGAGCGGCUACGUUAACAACCCCGAGGCUACAAACGCUCUCAUCGACAAGGACGGCUGGCUGCACAGCGGCGACAUCGCCUACUGGGACGAGGACGAGCACUUCUUCAUCGUGGACCGGCUGAAGAGCCUGAUCAAAUACAAGGGCUACCAGGUAGCCCCAGCCGAACUGGAGAGCAUCCUGCUGCAACACCCCAACAUCUUCGACGCCGGGGUCGCCGGCCUGCCCGACGACGAUGCCGGCGAGCUGCCCGCCGCAGUCGUCGUGCUGGAACACGGUAAAACCAUGACCGAGAAGGAGAUCGUGGACUAUGUGGCCAGCCAGGUUACAACCGCCAAGAAGCUGCGCGGUGGUGUUGUGUUCGUGGACGAGGUGCCUAAAGGACUGACCGGCAAGUUGGACGCCCGCAAGAUCCGCGAGAUUCUCAUUAAGGCCAAGAAGGGCGGCAAGAUCGCCGU GUAAYHuman GALT mRNA: (SEQ ID NO: 2)XAUGUCGCGCAGUGGAACCGAUCCUCAGCAACGCCAGCAGGCGUCAGAGGCGGACGCCGCAGCAGCAACCUUCCGGGCAAACGACCAUCAGCAUAUCCGCUACAACCCGCUGCAGGAUGAGUGGGUGCUGGUGUCAGCUCACCGCAUGAAGCGGCCCUGGCAGGGUCAAGUGGAGCCCCAGCUUCUGAAGACAGUGCCCCGCCAUGACCCUCUCAACCCUCUGUGUCCUGGGGCCAUCCGAGCCAACGGAGAGGUGAAUCCCCAGUACGAUAGCACCUUCCUGUUUGACAACGACUUCCCAGCUCUGCAGCCUGAUGCCCCCAGUCCAGGACCCAGUGAUCAUCCCCUUUUCCAAGCAAAGUCUGCUCGAGGAGUCUGUAAGGUCAUGUGCUUCCACCCCUGGUCGGAUGUAACGCUGCCACUCAUGUCGGUCCCUGAGAUCCGGGCUGUUGUUGAUGCAUGGGCCUCAGUCACAGAGGAGCUGGGUGCCCAGUACCCUUGGGUGCAGAUCUUUGAAAACAAAGGUGCCAUGAUGGGCUGUUCUAACCCCCACCCCCACUGCCAGGUAUGGGCCAGCAGUUUCCUGCCAGAUAUUGCCCAGCGUGAGGAGCGAUCUCAGCAGGCCUAUAAGAGUCAGCAUGGAGAGCCCCUGCUAAUGGAGUACAGCCGCCAGGAGCUACUCAGGAAGGAACGUCUGGUCCUAACCAGUGAGCACUGGUUAGUACUGGUCCCCUUCUGGGCAACAUGGCCCUACCAGACACUGCUGCUGCCCCGUCGGCAUGUGCGGCGGCUACCUGAGCUGACCCCUGCUGAGCGUGAUGAUCUAGCCUCCAUCAUGAAGAAGCUCUUGACCAAGUAUGACAACCUCUUUGAGACGUCCUUUCCCUACUCCAUGGGCUGGCAUGGGGCUCCCACAGGAUCAGAGGCUGGGGCCAACUGGAACCAUUGGCAGCUGCACGCUCAUUACUACCCUCCGCUCCUGCGCUCUGCCACUGUCCGGAAAUUCAUGGUUGGCUACGAAAUGCUUGCUCAGGCUCAGAGGGACCUCACCCCUGAGCAGGCUGCAGAGAGACUAAGGGCACUUCCUGAGGUUCAUUACCACCUGGGGCAGAAGGACAGGGAGACAGCAACCAUCGCCUGAY Human EPO mRNA:(SEQ ID NO: 3) XAUGGGGGUGCACGAAUGUCCUGCCUGGCUGUGGCUUCUCCUGUCCCUGCUGUCGCUCCCUCUGGGCCUCCCAGUCCUGGGCGCCCCACCACGCCUCAUCUGUGACAGCCGAGUCCUGGAGAGGUACCUCUUGGAGGCCAAGGAGGCCGAGAAUAUCACGACGGGCUGUGCUGAACACUGCAGCUUGAAUGAGAAUAUCACUGUCCCAGACACCAAAGUUAAUUUCUAUGCCUGGAAGAGGAUGGAGGUCGGGCAGCAGGCCGUAGAAGUCUGGCAGGGCCUGGCCCUGCUGUCGGAAGCUGUCCUGCGGGGCCAGGCCCUGUUGGUCAACUCUUCCCAGCCGUGGGAGCCCCUGCAGCUGCAUGUGGAUAAAGCCGUCAGUGGCCUUCGCAGCCUCACCACUCUGCUUCGGGCUCUGGGAGCCCAGAAGGAAGCCAUCUCCCCUCCAGAUGCGGCCUCAGCUGCUCCACUCCGAACAAUCACUGCUGACACUUUCCGCAAACUCUUCCGAGUCUACUCCAAUUUCCUCCGGGGAAAGCUGAAGCUGUACACAGGGGAGGCCUGCAGGACAGGGGACAGAUGAY 5′ and 3′ UTR Sequences(SEQ ID NO: 4) X = GGGAUCCUACC or (SEQ ID NO: 5)GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACG (SEQ ID NO: 6) Y = UUUGAAUU  or(SEQ ID NO: 7) CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC

Exemplary Formulation Protocol

Lipid nanoparticles (LNP) were formed via standard ethanol injectionmethods (Ponsa, M.; Foradada, M.; Estelrich, J. “Liposomes obtained bythe ethanol injection method” Int. J. Pharm. 1993, 95, 51-56). Ethanolicstock solutions of the lipids were prepared ahead of time at 50 mg/mLand stored at −20° C. FFL mRNA was stored in water at a finalconcentration of 1 mg/mL at −80° C. until the time of use. All mRNAconcentrations were determined via the Ribogreen assay (Invitrogen).Encapsulation of mRNA was calculated by performing the Ribogreen assaywith and without the presence of 0.1% Triton-X 100. Particle sizes(dynamic light scattering (DLS)) and zeta potentials were determinedusing a Malvern Zetasizer instrument in 1×PBS and 1 mM KCl solutions,respectively.

Formulation Example 1

Aliquots of 50 mg/mL ethanolic solutions of the imidazole-based cationiclipid ICE, DOPE and DMG-PEG2K were mixed and diluted with ethanol to 3mL final volume. Separately, an aqueous buffered solution (10 mMcitrate/150 mM NaCl, pH 4.5) of FFL mRNA was prepared from a 1 mg/mLstock. The lipid solution was injected rapidly into the aqueous mRNAsolution and shaken to yield a final suspension in 20% ethanol. Theresulting nanoparticle suspension was filtered, diafiltrated with 1×PBS(pH 7.4), concentrated and stored at 2-8° C. Final concentration=1.73mg/mL CO-FF mRNA (encapsulated). Z_(ave)=68.0 nm (Dv₍₅₀₎=41.8 nm;Dv₍₉₀₎=78.0 nm). Zeta potential=+25.7 mV.

Formulation Example 2

Aliquots of 50 mg/mL ethanolic solutions of DLinKC2DMA, DOPE,cholesterol and DMG-PEG2K were mixed and diluted with ethanol to 3 mLfinal volume. Separately, an aqueous buffered solution (10 mM acetate,pH 6.5) of FFL mRNA was prepared from a 1 mg/mL stock. The lipidsolution was injected rapidly into the aqueous mRNA solution and shakento yield a final suspension in 20% ethanol. The resulting nanoparticlesuspension was filtered, diafiltrated with 1×PBS (pH 7.4), concentratedand stored at 2-8° C. Final concentration=3.47 mg/mL CO-FF mRNA(encapsulated). Z_(ave)=74.3 nm (Dv₍₅₀₎=58.6 nm; Dv₍₉₀₎=95.2 nm).

All formulations were made in accordance to the procedure described inFormulation Example 1, with the exception of DLinKC2DMA formulations,which were formulated according to Formulation Example 2. Variousexemplary lipid nanoparticle formulation, are disclosed in Table 1. Alllipid ratios are calculated as mol percentage.

TABLE 1 Exemplary Lipid Nanoparticle Formulations. Lipid Ratio N/P LipidFormulation (Total Component) (mol %) Ratio C12-200:DOPE:CHOL:DMGPEG2K40:30:25:5 4 C12-200:DOPE:CHOL:DMGPEG2K 40:30:20:10 4C12-200:DOPE:CHOL:DMGPEG2K 40:30:20:10 2 C12-200:DOPE:CHOL:DMGPEG2K25:35:30:10 2 DLin-KC2-DMA:DOPE:CHOL:DMGPEG2K 50:25:20:5 5DLin-KC2-DMA:DOPE:CHOL:DMGPEG2K 50:20:20:10 5 HGT4003:DOPE:Chol:DMGPEG2K70:10:10:10 5 HGT4003:DOPE:Chol:DMGPEG2K 25:35:30:10 5HGT4003:DOPE:Chol:DMGPEG2K 50:25:20:5 5 HGT4003:DOPE:Chol:DMGPEG2K40:30:20:10 5 ICE:DOPE:DMGPEG2K 90:5:5 16  ICE:DOPE:DMGPEG2K 70:20:1016  ICE:DOPE:DMGPEG2K 70:25:5 16  ICE:DOPE:DMGPEG2K 90:5:5 8ICE:DOPE:DMGPEG2K 70:20:10 8 ICE:DOPE:DMGPEG2K 70:25:5 8DODAP:DOPE:Chol:DMGPEG2K 18:57:20:5 4 DODAP:DOPE:Chol:DMGPEG2K18:56:20:6 4 DODAP:DOPE:Chol:DMGPEG2K 18:52:20:10 4DLin-KC2-DMA:C12-200:DOPE:CHOL:DMGPEG2K 30:20:25:20:5 5C12-200:DOPE:ICE:DMGPEG2K 40:30:20:10 4 C12-200:DOPE:ICE:DMGPEG2K40:30:20:10 2 C12-200:ICE:DMGPEG2K 20:70:10 4 DODAP:DOPE:ICE:DMGPEG2K18:57:20:5 4 DODAP:DOPE:ICE:DMGPEG2K 18:37:40:5 4

“Blended” Formulations:

A portion of one cationic lipid formulation was combined with a separatealiquot of a different cationic lipid formulation in a desired ratio,based on encapsulated mRNA concentrations and dosed accordingly.

“Mixed” Formulations:

A single formulation synthesized from a previously combined organicsolution of helper lipids, PEGylated lipids and multiple, non-identicalcationic/ionizable lipids.

As used herein, the term “blend” refers to a combination of two or moreseparate, non-identical formulations. Typically, the two or moreseparate, non-identical formulations are combined or blended into onecomposition, such as, a suspension, as depicted, for example, in FIG. 1.As used herein, non-identical formulations refer to formulationscontaining at least one distinct lipid component. In some embodiments,non-identical formulations suitable for blend contain at least onedistinct cationic lipid component. The term “blend” as used herein isdistinguishable from the terms “mix” or “mixture”, which are used hereinto define a single formulation containing multiple non-identicalcationic/ionizable lipids, multiple non-identical helper lipids, and/ormultiple non-identical PEGylated lipids. In some embodiments, a “mix”formulation contains at least two or more non-identicalcationic/ionizable lipids. Typically, a “mix” formulation contains asingle homogeneous population of lipid nanoparticles.

Example 2. Injection Protocol and Assays for Expression andBiodistribution In Vivo Injection Protocol

All studies were performed using male or female CD-1 mice ofapproximately 6-8 weeks of age at the beginning of each experiment.Samples were introduced by a single bolus tail-vein injection orintracerebroventricular (ICV) administration of an equivalent total doseof encapsulated FFL mRNA up to a dose of 230 micrograms. Four hourspost-injection the mice were sacrificed and perfused with saline.

Isolation of Organ Tissues for Analysis

The liver, spleen and when applicable, the brain, of each mouse washarvested, apportioned into two parts and stored in either: (1)—10%neutral buffered formalin or; (2)—snap-frozen and stored at −80° C. forbioluminescence analysis.

Bioluminescence Assay Tissue Homogenization

The bioluminescence assay was conducted using a Promega Luciferase AssaySystem (Promega). Tissue preparation was performed as follows: briefly,portions of the desired tissue sample (snap-frozen) were thawed, washedwith DI water and placed in a ceramic bead homogenization tube. Thetissue was treated with lysis buffer and homogenized. Upon subjection tofive freeze/thaw cycles followed by centrifugation at 4° C., thesupernatant was transferred to a new microcentrifuge tube and stored at−80° C.

Luciferase Assay

The Luciferase Assay Reagent was prepared by adding 10 mL of LuciferaseAssay Buffer to Luciferase Assay Substrate and mixed via vortex. Twentymicroliters of each homogenate was loaded onto a 96-well plate followed,along with 20 microliters of plate control. Separately, 120 microlitersof Luciferase Assay Reagent was loaded into each well of a 96-well flatbottomed plate and analyzed using a Biotek Synergy 2 instrument tomeasure luminescence (measurements were recorded in relative light units(RLU)).

EPO Assay

Human EPO protein was detected via hEPO ELISA system (R&D Systems).Western blot analyses were performed using an anti-hEPO antibody MAB2871(R&D Systems) and ultrapure human EPO protein (R&D Systems) as acontrol.

Example 3. Delivery of CO-FFL mRNA Via Lipid-Derived Nanoparticles

For the study, animals were injected intravenously with a single dose ofencapsulated mRNA and sacrificed after four hours. Activity of expressedfirefly luciferase protein in livers and spleens was determined using abioluminescence assay. Detectable signal over baseline was observed forevery animal tested to determine the expression of firefly luciferaseprotein from the exogenous mRNA.

As illustrated in FIG. 2, all formulations tested yielded an enhancedluminescence output with respect to different controls (e.g., non-FFLmRNA encapsulated lipid nanoparticles, empty nanoparticles and PBS). Adetailed representation of the raw values of luminescence output(expressed as the median RLU/mg of total protein) from fireflyluciferase protein detected in the livers of mice 4 hours following theadministration of a single dose of the lipid formulations is presentedin Table 2 below. The controls used in the present study included aC12-200-based cationic lipid nanoparticle encapsulating anon-fluorescent mRNA (30 μg dose) and PBS vehicle.

TABLE 2 Luminescence Output of CO-FFL Protein in Mice Liver Dose of MeanLuminescent Lipid Formulation Encapsulated Output (Cationic Component)CO-FFL mRNA (ug) (RLU/mg protein) C12-200 30 2,770,000 DLin-KC2-DMA 902,280,000 HGT4003 200 2,420,000 ICE 200 557,000 DODAP 200 14,000*C12-200 (Control) 30 500 PBS (Control/No lipid) — 100 *C12-200control - Is a C12-200-based cationic lipid nanoparticle encapsulated 30ug of mRNA encoding a non-fluorescent protein — = No lipid control

Example 4. Blending of Two Separate, Non-Identical Lipid Nanoparticles,Leads to Synergistic Enhancement of Expression

In order to evaluate the transfection efficiency of various lipidencapsulating formulation of CO-FFL mRNA, both mixtures and blends ofvarious lipid nanoparticle formulations, as well as the individualconstituent lipid nanoparticles were prepared and assayed for theirability to transfect and express mRNA in various targeted cells andtissues (as determined by the luminescence of the firefly luciferaseprotein) in vivo.

Specifically, two lipid nanoparticle formulations encapsulating FFL mRNAand comprising either C12-200 or DLin-KC2-DMA as the cationic lipid wereprepared in accordance with the formulation protocol described inExample 1 (such formulations being referred to herein as Formulations 1and 2, respectively). A third lipid nanoparticle formulationencapsulating FFL mRNA was prepared which comprised a mixture of thecationic lipids C12-200 and DLin-KC2-DMA in a single lipid nanoparticle(referred to herein as Formulation 3). Finally, a fourth lipidnanoparticle formulation was prepared which comprised a blend ofFormulations 1 and 2 in a 1:3 ratio based on the dose of encapsulatedFFL mRNA (referred to herein as Formulation 4). Table 3 represents thecationic lipid component(s) of Formulations 1, 2, 3 and 4, as well asthe total dose of encapsulated FFL mRNA.

TABLE 3 Liver Fluorescence Intensity for Single, Mixed and Blended LipidFormulations Dose of Mean Encapsulated Luminescent Lipid FFL mRNA OutputFormulation (Cationic Component) (ug) (RLU/mg protein) #1 C12-200 302,770,000 #2 DLin-KC2-DMA 90 2,280,000 #3 C12-200/DLin-KC2- 30 1,530,000DMA “Mix” #4 “Blend” of Formulations 120  46,800,000  #1 and #3 Rawvalues of mean luminescence output from FFL protein in livers of miceafter treatment with FFL mRNA-loaded lipid nanoparticles demonstratingthe difference between a “mixed” formulation versus a “blended”formulation. Formulation #3 represents a single formulation of “mixed”cationic lipids (30 ug dose) while formulation #4 represents a “blend”of formulations #1 and #2 as a 1:3 ratio (based on dose of encapsulatedmRNA). Values are depicted as mean RLU/mg of total protein in liver fourhours post-administration.

Animals were injected intravenously (via a tail-vein injection) with asingle dose of FFL mRNA encapsulated in either Formulations 1, 2, 3 or 4and sacrificed after four hours. The activity of expressed fireflyluciferase mRNA in the livers and spleens of the animals was determinedin a bioluminescence assay. A detectable signal over baseline wasobserved in every animal tested, inferring the expression theexogenously-administered encapsulated FFL mRNA and the correspondingproduction of the firefly luciferase protein.

A comparison of the luminescence output from FFL protein expressed inthe liver upon delivery via various liposomal nanoparticles wasevaluated. Luminescence from a single formulation varied in intensitybased upon which cationic lipid is employed. Such luminescence can alsobe dependent on (but not exclusively) lipid composition, total lipidcontent and dose. All formulations tested, however, yielded an enhancedlight output with respect to different controls (non-FFL mRNA loadednanoparticles, empty nanoparticles, PBS, etc.) (FIG. 2). A more detailedrepresentation of the luminescent output (raw values) of theseformulations is listed in Table 3.

As shown in Table 3, mixing different cationic lipids within a singleformulation (C12-200, DLin-KC2-DMA) still allows for successful deliveryof the desired mRNA to the target tissue with comparable overallproduction of protein (based on measured RLU output of FFL protein) ascompared to either C12-200 or DLin-KC2-DMA based formulation. However,upon blending two separate, non-identical formulations, a synergistic(non-additive) enhancement in light output was observed (FIG. 3, Table3). Specifically, as listed in Table 3, when blending a C12-200-basedlipid nanoparticle encapsulating FFL mRNA (Formulation #1) with aDLin-KC2-DMA-based FFL mRNA-loaded lipid nanoparticle (Formulation #2)in a 1:3 ratio (30 ug FFL mRNA:90 ug FFL mRNA, respectively), oneobserves a mean RLU value of 46.8×10⁶ RLU/mg protein (Formulation #4).This is compared to an expected additive value of 5.05×10⁶ RLU/mgprotein, based on the sum of each formulation tested individually(2.77×10⁶ and 2.28×10⁶ RLU/mg protein for Formulation #1 and #2,respectively (FIG. 3, Table 3)). By administering a blend of the twoformulations, one achieves 9-fold greater luminescent output (i.e. moreefficacious production of desired protein).

On the contrary, simply mixing two lipids within one lipid nanoparticledid not result in any observable enhancement (Formulation #3, Table 3).For example, a 30 ug dose of a mixed C12-200/DLin-KC2-DMA FFLmRNA-loaded lipid nanoparticle yielded a comparable, albeit lowerluminescent output than either a C12-200 or DLin-KC2-DMA FFL mRNA-loadedlipid nanoparticle alone (1.53×10⁶ RLU/mg protein vs. 2.77×10⁶ and2.28×10⁶ RLU/mg protein, respectively) (FIG. 3, Table 3). When dosingsuch a “mix” at 120 ug, the resulting formulation was lethal. It isnoteworthy that blending two separate formulations at this dose (120 ug)was well tolerated in mice after 4 hours.

The effect of mixing, was further evaluated using two additional CO-FFLmRNA lipid mixtures: C12-200/ICE FFL and DODAP/ICE FFL. When theC12-200/ICE FFL mixed formulation was dosed at 30 ug, the resulting meanluminescent output detected was 2.71×10⁶ RLU/mg protein, comparable toan individual C12-200-based lipid formulation (2.77×10⁶ RLU/mg protein).The DODAP/ICE FFL mixed formulation, resulted in a mean output of 7,300RLU/mg protein as compared to a single DODAP-based formulation (˜14,000RLU/mg protein). As stated above, a mixed formulation has yieldedcomparable results to single cationic lipid-based formulations but notenhanced.

Example 5. Synergy Observed Across a Variety of Different LipidNanoparticle Blends

This example demonstrates that the synergy observed in Example 4 is notlimited to specific formulations blended. In fact, this synergy isobserved across a wide variety of different lipid nanoparticles blendedin various ratios. Exemplary results from various experiments aresummarized in Table 4.

Specifically, multiple lipid nanoparticle formulations encapsulating FFLmRNA and comprising either C12-200, DLin-KC2-DMA, ICE, DODAP or HGT4003as the cationic lipid were prepared in accordance with the formulationprotocol described in Example 1, and subsequently blended at variousratios to prepare the blended lipid nanoparticle formulation (Table 4).In addition, the lipid formulations designated as “(A)” comprise a 5%concentration of the PEG-modified lipid DMG-PEG2000, while the lipidformulations designated as “(B)” comprise a 10% DMG-PEG2000. Animalswere injected intravenously (via a tail-vein injection) with a singlebolus dose of FFL mRNA encapsulated in either a blended lipidformulation of the constituent lipid formulation and sacrificed afterfour hours. The activity of expressed firefly luciferase mRNA in theliver was determined in a bioluminescence assay. Increases range from1.1-10×luminescence over the sum of each respective individualformulation demonstrating a synergistic enhancement.

As shown in Table 4, each blend resulted in an increase in luminescencewhen delivering FFL mRNA as compared to the sum of the output of itsindividual formulations. Experiments #1-4 (Table 4) were similar to whathas been described above (Example 4). The results shown in Table 4 alsoindicate that upon varying the formulation parameters (PEG percentage)for each nanoparticle, one can effectively adjust and control the amountof synergistic enhancement. Further, this synergistic enhancement inprotein production can be impacted/tailored by lipid composition (otherthan PEGylated lipid) and total lipid content.

Another example of synergistic enhancement using different cationiclipid formulations is represented by Experiment #8. When blending a FFLmRNA-loaded HGT4003-based lipid nanoparticle with a FFL mRNA-loadedICE-based lipid nanoparticle in a 1:1 ratio (100 ug encapsulated mRNA:100 ug encapsulated mRNA, respectively), one observes a mean RLU/mgprotein value of 4.39×10⁵. This is compared to an expected additivevalue of 2.77×10⁵ RLU/mg protein, based on the sum of each formulationtested individually ((2.53×10⁵ and 2.40×10⁴ RLU/mg protein, respectively(Table 4, FIG. 4)).

Another factor is the ratio at which the formulations are blended. Asdepicted in FIG. 4, two formulations encapsulating FFL mRNA are dosedindividually and as blends employing two different blend ratios, 1:1 and3:1 (HGT4003:ICE) (Table 4, Experiment #8 and #9, respectively). While asynergistic increase in luminescence is observed for both blends, oneobserves a much greater enhancement when the two formulations areblended in a 3:1 ratio (HGT4003:ICE). The formulations blended in a 1:1ratio yielded a 1.59-fold enhancement over the sum of its individualcounterparts, while the formulations blended in a 3:1 ratio afforded anapproximate 4.4-fold enhancement.

The results described herein demonstrate that blending two separate FFLmRNA-loaded lipid nanoparticles mechanistically allows the production ofmore FFL protein to be produced within the mouse liver in a synergisticfashion than as compared to its separate counterparts. Without wishingto be bound by any theory, it is contemplated that possible explanationsfor synergy include: a.) non-competing pathways of cellular entry, b.)combination of different intracellular trafficking mechanisms(proton-sponge vs. fusogenicity, c.) “endosomal fusion” combining drugrelease properties with endosomal release properties (i.e., a cell cantake up both separate nanoparticles and as they get processed throughthe endosomal pathway, the endosomes fuse and then one set of lipidsenhances the other in terms of mRNA release), d.) modulation of activeinhibitory pathways allowing greater uptake of nanoparticles.

TABLE 4 Various cationic lipid-nanoparticle blends and synergisticenhancement of CO-FFL protein luminescence in liver Lipid Formulation #1RLU/mg protein Lipid Formulation #2 RLU/mg protein Total Dose RLU/mgprotein Fold Increase in Experiment (FFL mRNA) of Formulation #1 (FFLmRNA) of Formulation #2 Ratio (ug mRNA) of Blend Luminescence 1 C12-200(A) 200,526 DLin-KC2-DMA (A) 47,231 1:3 120 775,275 3.12 2 C12-200 (A)1,299,758 DLin-KC2-DMA (B) 26,661 1:3 120 2,523,496 1.90 3 C12-200 (B)396,134 DLin-KC2-DMA (A) 47,231 1:3 120 1,766,473 3.98 4 C12-200(B)396,134 DLin-KC2-DMA (B) 26,661 1:3 120 1,673,159 3.96 5 C12-200 (A)1,062,038 ICE —^(a) 1:6.67 230 4,325,041 3.98 6 C12-200 (A) 2,101,685DODAP 12,000^(c) 1:3 120 1,985,508 0 7 DLin-KC2-DMA (A) 1,634,139 DODAP12,000^(c) 3:1 120 2,650,865 1.61 8 HGT4003 252,884 ICE 24,134 1:1 200439,228 1.59 9 HGT4003 —^(a) ICE —^(a) 3:1 200 1,225,835 4.43^(d) 10 ICE24,134 DODAP 7,649 1:1 200 105,230 3.31 Summary of synergisticenhancement of FFL protein luminescence in livers of treated mice (N =4) four hours post-administration when blending two separate fluorescentmRNA-loaded lipid nanoparticles (FFL mRNA). Increases range from 1.1-10xluminescence over the sum of each respective individual formulationdemonstrating a synergistic enhancement. The lipids listed represent thecationic lipid component of each formulation. A formulation of (A)consists of 5% DMGPEG2K while (B) incorporates 10% DMGPEG2K in theformulation. Values are representative of observable enhancement fourhours post-administration. ^(a)Not dosed as separate formulation thisexperiment. ^(b)Fold increase determined using average value for ICEformulation previously measured. ^(c)Historical data luminescence fromprevious experiment. ^(d)Fold increase based on comparison of Experiment8 as 1:1 ratio.

Example 6. Synergistic Effect is Independent of Nucleic AcidIncorporated

This synergistic enhancement of light production is even more evidentwhen substituting non-fluorescent “dummy” mRNA (non-FFL) lipidnanoparticles as a blend component. Specifically, representativeexperiments incorporating mRNA encoding galactose-1-phosphate uridyltransferase (GALT) as the non-fluorescent component were performed todemonstrate the notion of synergy with respect to blending separateformulations is independent of the nucleic acid incorporated (FIG. 5).

FIG. 5 illustrates a comparison of the median luminescence observed whenstudying blended formulations encapsulating fluorescent (FFL) andnon-fluorescent (GALT) mRNA. The C12-200-based lipid nanoparticleformulations were administered at a dose of 30 μg of mRNA, while theDLin-KC2-DMA-based lipid nanoparticle formulations were administered ata dose of 90 μg of mRNA. The blended formulations were both administeredat doses of 120 μg total mRNA. As illustrated in FIG. 5, an enhancedmedian luminescence was observed in both blended formulations relativeto the luminescence observed when individually administering theconstituent lipid nanoparticles.

Table 5 lists some representative examples of various cationiclipid-based systems which demonstrate a synergistic production of FFLprotein when incorporating a non-fluorescent GALT message in one of theformulations.

As can be seen in Table 5, the synergy is evident across a range ofcationic lipids employed (C12-200, DLin-KC2-DMA, HGT4003, ICE, DODAP,etc., Table 5). In some instances, one observes enhancements of over30-fold higher than the individual formulations administered separately(Table 5, Experiment #14). This was evident when blending a formulationof the disulfide-based lipid HGT4003 encapsulating FFL mRNA with aC12-200-based formulation encapsulating GALT mRNA (Table 5, Experiment#17). The observed mean RLU measured in the livers of mice four hourspost-administration was 8.38×106 RLU for the blend as compared to3.85×10⁵ RLU from the HGT4003-based FFL mRNA-loaded nanoparticleindependently. Negligible background fluorescence was observed for allnon-fluorescent GALT mRNA loaded lipid nanoparticle groups.

Another example of observed synergistic enhancement when blending a FFLmRNA-loaded lipid nanoparticle with a GALT mRNA-loaded lipidnanoparticle is represented in Experiment #18 (Table 5). A blend of aFFL mRNA-loaded ICE-based lipid nanoparticle with a non-fluorescent GALTmRNA-loaded DLin-KC2-DMA-based lipid nanoparticle (1:1 ratio based ondose of encapsulated mRNA) yielded an observed mean RLU/mg protein valueof 1.84×10⁵. This is compared to a value of 3.76×10⁴ RLU/mg protein forthe FFL mRNA-loaded ICE-based formulation administered independently.Such a blend afforded an approximate 4.85-fold enhancement inluminescent output.

Thus, experiments shown in Table 5 clearly demonstrate that synergisticenhancement with respect to light production does not dependent on bothnanoparticles to have a fluorescent message incorporated, suggestingthat synergistic effects with respect to blending separate lipidformulations is independent of the incorporated nucleic acid.

TABLE 5 Various cationic lipid-nanoparticle blends and synergisticenhancement of CO-FFL protein luminescence in liver Lipid Formulation #1RLU/mg protein Lipid Formulation #2 Total Dose RLU/mg protein FoldIncrease in Experiment (FFL mRNA) of Formulation #1 (GALT mRNA) Ratio(ug mRNA) of Blend Luminescence 11 C12-200 6,866,021 DLin-KC2-DMA 1:3120 23,752,692 3.46 12 C12-200 32,560,660 HGT4003 1:3.33 130 51,323,2771.58 13 DLin-KC2-DMA 4,983,048 C12-200 3:1 120 22,124,581 4.41 14DLin-KC2-DMA 255,921 ICE 1:1 200 7,711,698 30.10 15 DLin-KC2-DMA1,382,039 HGT4003 1:1 120 1,555,411 1.13 16 HGT4003 137,068 DLin-KC2-DMA1:1 120 305,285 2.22 17 HGT4003 385,110 C12-200 3.33:1 130 8,376,00021.72 18 ICE 37,571 DLin-KC2-DMA 1:1 200 184,322 4.85 19 ICE 68,562DODAP 1:1 200 69,480 1.01 20 DODAP 8,425 ICE 1:1 200 18,736 2.20 21C12-200 50,500,335 DODAP 1:5 150 29,515,643 0 22 DODAP 25,745 C12-2005:1 150 22,064,370 857 Summary of synergistic enhancement of FFL proteinluminescence in livers of treated mice (N = 4) four hourspost-administration when blending a fluorescent mRNA-loaded lipidnanoparticle (FFL mRNA) with a non-fluorescent mRNA-loaded lipidnanoparticle (GALT mRNA). Increases range from 0-30x luminescence overthe single formulation demonstrating a synergistic enhancement. Theratios listed are representative of the fluorescent formulation dose:non-fluorescent formulation dose. The lipids listed represent thecationic lipid component of each formulation. Values are based onobservable enhancement four hours post-administration.

We then tested if the synergistic effect with respect to the blending ofdifferent lipid formulations dependents on the presence of messengerRNA. To that end, empty liposomal nanoparticles (without any mRNAencapsulated) were tested as possible synergistic agents. Interestingly,blending an empty C12-200-based cationic lipid nanoparticle with a FFLmRNA-loaded DLin-KC2-DMA-based cationic lipid nanoparticle resulted in astrong synergistic enhancement of luminescence (4-fold increase) (FIG.6, Table 6, Experiment #24) while the respective inverse (FFLmRNA-loaded C12-200 nanoparticles with empty DLin-KC2-DMA liposomalnanoparticle) blend resulted in no enhancement (FIG. 6, Table 6(Experiment #23)).

These results indicate that the synergistic effect may be dependent onthe presence of message, at least in some cases. Without wishing to bebound by any particular theory, it is contemplated that two possiblelipid-specific mechanisms may be leading to synergistic effect with orwithout the presence of mRNA.

TABLE 6 Effect of mRNA encapsulation on blending enhancement RLU/mgTotal Lipid protein of Lipid Dose RLU/mg Fold Formulation #1 FormulationFormulation #2 (ug protein of Increase in Experiment (FFL mRNA) #1 (nomRNA) Ratio mRNA) Blend Luminescence 23 C12-200 6,407,934 DLin-KC2-DMA1:3 120 3,940,501 0 24 DLin-KC2-DMA 5,694,440 C12-200 3:1 120 23,272,2444.09

Example 7. Synergistic Effect of Liposome-Derived Nanoparticles Via ICVDelivery

As shown in the above examples, the synergistic increase in proteinproduction has been observed in the liver over multiple systems whentest articles have been administered intravenously as described (videsupra). It is contemplated that the synergistic effect may be appliedfurther, not only to diseases specific to the liver but anywhere one mayrequire treatment, i.e. lung, spleen, kidney, heart, eye, centralnervous system, brain, etc. To confirm that, the present exampledemonstrates a synergistic enhancement of FFL protein luminescence whendelivering a blend of two separate FFL mRNA-loaded lipid nanoparticlesvia intracerebroventricular (ICV) administration.

As shown in FIG. 7, C12-200-based FFL mRNA-encapsulated lipidnanoparticles blended with FFL mRNA-loaded DLin-KC2-DMA-based lipidnanoparticles in a 1:3 ratio (based on mRNA dose) resulted in anapproximate 2.0-fold enhancement of FFL protein luminescence as comparedto the sum of the luminescent output of each individual formulation(1.11×10⁵ RLU vs 5.57×10⁴ RLU, respectively). To evaluate whether theblended formulations were capable of demonstrating a synergisticenhancement of observed luminescence in the cells and tissues of thecentral nervous system, additional studies were conducted whereinblended formulations were administered to animals via theintracerebroventricular (ICV) route of administration. In particular,the synergistic enhancement in the expression of ICV-administeredexogenous mRNA encapsulated in a blended lipid nanoparticle formulationand the corresponding production of the firefly luciferase proteinencoded thereby (as demonstrated by the enhanced median luminescentoutput) was evaluated by comparing median luminescence output observedfollowing ICV administration of the blended lipid formulations to themedian luminescence observed output observed following ICVadministration of the individual constituent lipid nanoparticleformulations.

Example 8. Synergistically Enhancing the Expression of Secreted Proteins

This example demonstrates that the synergistic phenomenon also appliestowards the idea of a “depot” effect for secretion of a desired proteininto the bloodstream. For example, the delivery of mRNA encoding humanerythropoietin (EPO) can be packaged via a lipid nanoparticle andinjected into a mouse. The formulation can accumulate within the liverand/or other organs and transcribe the mRNA to the desired EPO protein.Upon its expression, the protein can secrete from the liver (organ) andfunction as necessary.

This notion was tested using a human EPO mRNA-loaded C12-200-based lipidnanoparticle, a human EPO mRNA-loaded DLin-KC2-DMA-based lipidnanoparticle and a blend of the two formulations in various ratios.Fully secreted human EPO was detected via ELISA and immunoblot analyses(FIGS. 8 and 9, respectively) four hours after intravenousadministration of these nanoparticles in mice. Further, upon treatmentwith a blend of the two single formulations, one observed a synergisticenhancement of human EPO protein secreted into the bloodstream ascompared to the sum of each individual formulation counterpart (FIG. 8).This enhancement was more pronounced upon blending at a ratio of 1:3(˜2-fold) as compared to 1:1 (˜1.4 fold) (C12-200-basedformulation:DLin-KC2-DMA-based formulation).

This synergistic augmentation of protein production is more pronouncedwhen analyzed via western blot. As depicted in FIG. 9, a blend of ahuman EPO mRNA-loaded C12-200-based lipid nanoparticle with an analogousDLin-KC2-DMA-loaded lipid nanoparticle shows a strong band (Lane 4)significant of human EPO protein isolated from the serum four hourspost-administration. The lane corresponding to an individual C12-200formulation (Lane 2) yields a moderately detected band while that of theDLin-KC2-DMA-based formulation (Lane 3) is undetectable. One canqualitatively observe an enhancement of protein production as comparedto the “additive total” from each individual formulation.

These observations confirm that this synergistic phenomenon is notspecific to a luciferase system, but can be applicable to other targetproteins generally. In addition, as demonstrated in Example 7, thesynergistic phenomenon is observed not only via intravenous delivery tothe liver, but also via intracerebroventricular delivery to the brain.In addition to delivering to specific target organs, the blending of twoformulations can synergistically enhance the production of secretedproteins as demonstrated with our human EPO system.

The synergistic enhancement presented here suggests that equivalenttherapeutic efficacy can be achieved via administration of asignificantly lower dose than previously anticipated. The ability tocreate a synergistic production of protein via lipid-based nanoparticledelivery of mRNA allows for a much greater therapeutic window for thetreatment of a host of diseases. The successful delivery of such mRNA tothe liver and in particular, to hepatocytes, can be used for thetreatment and the correction of in-born errors of metabolism that arelocalized to the liver. Diseases such as ASD and OTC among other ureacycle disorders may be treated through mRNA therapy of the missing gene.Metabolic zonation of the urea cycle to hepatocytes means that providingthe missing enzyme in these cells should greatly improve normalbiochemical processing in individuals with these disorders. To achievethis in a synergistic fashion via the process described above, one couldadminister a much lower dose (˜2 to 30-fold lower) and achieve equal orgreater efficacy while mediating any adverse or toxic events.

1. A method of delivering a messenger RNA (mRNA) to a subject,comprising administering to the subject a pharmaceutical compositioncomprising an mRNA and a blend of at least a first lipid nanoparticleand a second lipid nanoparticle, wherein the first lipid nanoparticlecomprises the mRNA; and wherein the first lipid nanoparticle comprisesat least one lipid distinct from the second lipid nanoparticle.
 2. Themethod of claim 1, wherein the second lipid nanoparticle comprises anmRNA identical to the mRNA in the first lipid nanoparticle.
 3. Themethod of claim 1, wherein the second lipid nanoparticle comprises anmRNA not identical to the mRNA in the first lipid nanoparticle.
 4. Themethod of claim 1, wherein the at least one lipid distinct is cationiclipid.
 5. (canceled)
 6. The method of claim 1, wherein the first lipidnanoparticle and the second lipid nanoparticle comprise one or morehelper lipids.
 7. (canceled)
 8. The method of claim 1, wherein the firstlipid nanoparticle and the second lipid nanoparticle comprise one ormore PEG-modified lipids. 9-16. (canceled)
 17. The method of claim 1,wherein the mRNA is delivered to target cells selected from the groupconsisting of hepatocytes, epithelial cells, hematopoietic cells,epithelial cells, endothelial cells, lung cells, bone cells, stem cells,mesenchymal cells, neural cells, cardiac cells, adipocytes, vascularsmooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells,pituitary cells, synovial lining cells, ovarian cells, testicular cells,fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytesand tumor cells. 18-23. (canceled)
 24. The method of claim 1, whereinthe mRNA is selected from SEQ ID NO: 2 or SEQ ID NO:
 3. 25. The methodof claim 1, wherein the mRNA comprises a chemical modification. 26-32.(canceled)
 33. The method of claim 1, wherein the expression of the mRNAfollowing the administration of the pharmaceutical composition to thesubject exceeds the expression of the mRNA administered with the firstlipid nanoparticle but without the second lipid nanoparticle.
 34. Themethod of claim 33, wherein the expression of the mRNA following theadministration of the pharmaceutical composition to the subject exceedsthe expression of the mRNA administered with the first lipidnanoparticle but without the second lipid nanoparticle by at least abouttwo-fold.
 35. The method of claim 33, wherein the expression of the mRNAfollowing the administration of the pharmaceutical composition to thesubject exceeds the expression of the mRNA administered with the firstlipid nanoparticle but without the second lipid nanoparticle by at leastabout five-fold.
 36. The method of claim 33, wherein the expression ofthe mRNA following the administration of the pharmaceutical compositionto the subject exceeds the expression of the mRNA administered with thefirst lipid nanoparticle but without the second lipid nanoparticle by atleast about ten-fold.
 37. (canceled)
 38. The method of claim 1, whereinthe ratio of the first lipid nanoparticle to the second lipidnanoparticle in the pharmaceutical composition is about 1:1.
 39. Themethod of claim 1, wherein the ratio of the first lipid nanoparticle tothe second lipid nanoparticle in the pharmaceutical composition is about2:1.
 40. The method of claim 1, wherein the ratio of the first lipidnanoparticle to the second lipid nanoparticle in the pharmaceuticalcomposition is about 3:1.
 41. The method of claim 1, wherein the ratioof the first lipid nanoparticle to the second lipid nanoparticle in thepharmaceutical composition is about 4:1.
 42. A pharmaceuticalcomposition for delivery of messenger RNA (mRNA) polynucleotides to acell, the composition comprising a blend of a first lipid nanoparticleand a second lipid nanoparticle, wherein the first lipid nanoparticlecomprises the mRNA; and wherein the first lipid nanoparticle comprisesat least one lipid distinct from the second lipid nanoparticle. 43-68.(canceled)
 69. A method of delivering a messenger RNA (mRNA), comprisingadministering the mRNA formulated in at least two distinct lipidformulations.
 70. The method of claim 69, wherein the at least twodistinct lipid formulations differ in at least one cationic lipid.71-82. (canceled)